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Laryngeal squamous cell carcinoma (LSCC) with a high incidence and mortality rate, has a serious impact worldwide. Phosphofructokinase-1 liver type (PFKL) is a major enzyme in glycolysis progress, but its role in modulating tumorigenesis and cisplatin (DDP) chemosensitivity of LSCC was still unclear. The mRNA and protein levels of PFKL were detected by qRT-PCR and immunohistochemical assay. Cell Counting Kit-8 assay and flow cytometry were carried out to observe cell viability, as well as apoptosis and mitochondrial reactive oxygen species (mito-ROS). Extracellular acidification rate and lactate content were measured using extracellular flux analysis and lactate assay kit. Immunofluorescent staining was used to evaluate the expression of γ-H2AX foci. DNA damage was detected via single-cell gel electrophoresis. Western blotting was introduced to evaluate the protein level of PFKL, LDHA, γ-H2AX, cleaved PARP, H3K27ac, and H3K9ac. Mice xenograft model of LSCC was built for in vivo validation. The PFKL expression was significantly increased in LSCC and associated with poor survival of LSCC patients. Knockdown of PFKL in LSCC cells significantly inhibited cell viability, ECAR, lactate content, and LDHA expression, but promoted mito-ROS level. Furthermore, knockdown of PFKL enhanced response of LSCC cells to DDP by increasing DDP-induced apoptosis, promoting DDP-induced mito-ROS level, γ-H2AX foci, tail DNA, and the expression of γ-H2AX and cleaved PARP. However, the overexpression of PFKL in LSCC cells had opposite experimental results. Nude mice tumor formation experiment proved that downregulation of PFKL significantly enhanced response of cells to DDP, demonstrated by reduced tumor volume, weight and increased TUNEL-positive cells. Suppression of CBP/EP300-mediated PFKL transcription inhibited cell viability and glycolysis and promoted mito-ROS in LSCC. PFKL promotes cell viability and DNA damage repair in DDP-treated LSCC through regulation of glycolysis pathway., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare that are relevant to the content of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The sensitivity of laryngeal squamous cell carcinoma (LSCC) to chemotherapy shows large heterogeneity. The role of miRNA in small extracellular vesicles (sEV) in chemotherapy resistance is under investigation. However, the regulation and sorting mechanism of sEV miRNAs remains unclear. In this study, small RNA sequencing was used to explore miRNA expression profiles in sEV of LSCC after cisplatin stimulation; RNA pull-down, mass spectrometry, and EMSA were used to clarify the binding of candidate RNA-binding protein (RBP) and candidate miRNA. Immunostaining and microRNA fluorescence in situ hybridization were performed to identify how candidate RBP affects miRNA stability and nuclear/cytoplasmic distribution. In vivo experiments were performed to verify the biological functions and response to cisplatin of candidate RBP. We found that cisplatin stimulation induced increased expression of miR-148a-3p and sEV sorting. ANXA11 binds to miR-148a-3p in a sequence-specific manner. ANXA11 inhibits tumor cell proliferation and drug resistance by binding to and retaining miR-148a-3p. Cisplatin stimulation reduced ANXA11 expression and promoted miR-148a-3p efflux through sEV pathways. ANXA11 overexpression reduced in vivo tumor proliferation and cisplatin-resistance. Taken together, ANXA11 mediates cisplatin resistance through sEV miRNA resorting. Mechanically, ANXA11 binds to miR-148a-3p in a sequence-specific manner to regulate its resorting and thus influences tumor proliferation and chemoresistance., (© 2024 Federation of American Societies for Experimental Biology.)

Objective: This retrospective study analyzed the efficacy of PD-1 inhibitors combined with albumin-bound paclitaxel and cisplatin (TP regimen) in the treatment of recurrent and metastatic hypopharyngeal/laryngeal squamous cell carcinoma (RMHSCC/RMLSCC)., Methods: Patients diagnosed and treated at the Sun Yat-sen University Cancer Center from August 1, 2020, to August 15, 2023, with histologically confirmed RMHSCC/RMLSCC were included. All patients received PD-1 inhibitors combined with albumin-bound paclitaxel (260mg/m2) and cisplatin (60mg/m2) for 3-4 cycles. The primary endpoints were overall survival (OS) and progression-free survival (PFS)., Results: A total of 50 patients with RMHSCC/RMLSCC who received TP+PD-1 inhibitor therapy were included, with an objective response rate (ORR) of 56.0% (28/50). The 1-year and 2-year OS rates were 80.2% (95% CI: 69.3%-92.9%) and 68.6% (95% CI: 52.6%-89.5%), respectively, while the 1-year and 2-year PFS rates were 44.7% (95% CI: 31.9%-62.5%) and 26.0% (95% CI: 12.6%-53.4%), respectively. Treatment-related adverse events mainly included rash, myelosuppression, gastrointestinal reactions, and hypothyroidism., Conclusion: In the treatment of RMHSCC/RMLSCC with TP + PD-1 inhibitors, survival rates of patients can be improved while ensuring the safety of the treatment regimen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fang, Li, Xu, Cao, Wu, Zhang, Chen, Gao, Su and Liu.)

Background: Large cervical lymph nodes and the extranodal extension of metastatic lymph nodes are considered poor prognostic factors in head and neck squamous cell carcinoma (HNC)., Aims/objectives: The efficacy of intra-arterial chemotherapy (iaCT) targeting lymph node (LN) in HNC was examined., Materials and Methods: We performed a retrospective review of 41 patients with laryngeal and hypopharyngeal cancer showing metastatic cervical LN larger than 20 mm treated with iaCT with concurrent radiotherapy. The administration of cisplatin into LN was divided into three groups: no administration (NO), via the same artery as that supplying the primary tumor (SAME), and via a different artery from that supplying the primary tumor (DIFFERENT)., Results: A trend toward a more favorable three-year regional control in DIFFERENT compared to NO was observed, although the mean size of LN in DIFFERENT was larger than in the other groups. A better regional control was obtained in both DIFFERENT ( p  < .05) and DIFFERENT + SAME ( p  < .05) when overall rather than partial enhancement of lymph node by CT angiography was observed. Extranodal extension could be a factor predicting unfavorable regional control., Conclusions/significance: Targeting lymph node may be helpful to avoid neck dissection when iaCT was planned in HNC with relatively large LNs.

Chemotherapies such as 5-fluorouracil (5-FU) and cisplatin (CDDP) have been widely used to treat laryngeal squamous cell carcinoma (LSCC), the second most common head and neck squamous cell carcinoma. However, chemoresistance seriously impairs chemotherapeutic efficacy. Our present study reveals that 5-FU and CDDP treatment increase the expression of histone deacetylase 1 (HDAC1) in LSCC cells. Consistently, increased levels of HDAC1 are observed in chemoresistant cells. Knockdown of HDAC1 significantly restores the sensitivity of LSCC cells, as HDAC1 increases the expression of interleukin-8 (IL-8), which is essential for LSCC chemoresistance. Mechanistically, HDAC1 directly initiates the transcription of IL-8 though binding to its promoter. Simultaneously, si-HDAC1 increases the levels of miR-93, which binds to the 3'UTR of IL-8 mRNA to trigger its degradation. In summary, the HDAC1/IL-8 axis can confer chemotherapeutic resistance to LSCC cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: We aimed to explore the efficacy of low-temperature plasma ablation (LTPA) combined with low-dose cisplatin chemotherapy in laryngeal carcinoma patients and its influence on tumor markers., Methods: A total of 86 patients with laryngeal carcinoma were selected and divided into control group (n=43, treated with routine chemotherapy) and observation group (n=43, treated with LTPA combined with low-dose cisplatin chemotherapy) according to different treatment methods. The efficacy, immune function, adverse reactions and tumor marker levels were assessed in both groups, and the survival rate of patients was compared between the two groups., Results: The objective response rate and disease control rate in the observation group were significantly higher than those in the control group (p<0.05). After treatment, the observation group had higher levels of cluster of differentiation (CD)3+, CD4+ and CD4+/CD8+ than the control group, and a lower level of CD8+ than the control group (p<0.05). At 4, 8 and 16 weeks after treatment, the levels of serum carcinoembryonic antigen (CEA), carbohydrate antigen 19.9 (CA19.9) and CA125 declined significantly in both groups compared with those before treatment, more obviously in the observation group (p<0.05). Moreover, the incidence rate of postoperative complications in the observation group was evidently lower than that in the control group (p<0.05). According to the 5-year follow-up, the 5-year survival rate in the observation group was significantly higher than that in the control group (p<0.05)., Conclusions: LTPA combined with low-dose cisplatin chemotherapy has better efficacy in laryngeal carcinoma patients, and it can improve the patient's immune function with less toxic and side effects, which is worthy of clinical popularization and application.

circRNAs have been shown to be involved in cancer progression. It is unclear whether circPGAM1 exerts its effect on laryngocarcinoma drug resistance. In this study, we employed colony formation and MTT assay to determine colony number and cell viability under cisplatin treatment. TUNEL experiment was used to evaluate apoptosis of laryngocarcinoma cells in the presence of cisplatin. Xenograft tumor experiment was performed to assess in vivo tumor growth of SNU46 cells. We found that circPGAM1 enhanced colony formation and viability of SNU46 and M4E cells. In contrast, circPGAM1 caused attenuated cell apoptosis. Furthermore, we also confirmed that circPGAM1 played a key role in tumor growth in animal model and clinical patients. miR-376a was identified and proved to act as key effector for circPGAM1-mediated drug resistance. Finally, autophagy-related gene ATG2A was shown to rescue miR-376a-modulated drug resistance of laryngocarcinoma cells. Herein, we illuminate the role of circPGAM1 in laryngocarcinoma drug resistance, thereby facilitating development of targeted therapy for treating laryngocarcinoma., Competing Interests: Declaration of competing interest We declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This study describes a kinetic spectrophotometric method for accurate, sensitive and rapid determination of cisplatin in biofluids. The developed method is based on the inhibitory effect of cisplatin on the oxidization of Janus Green by bromate in acidic media. The change in absorbance as the criteria of the oxidation reaction was followed spectrophotometrically. To obtain the highest rate of sensitivity, efficient reaction parameters were optimized. Under optimum experimental conditions, a calibration graph was obtained linearly over the range 10.0 - 5750.0 μg L -1 and the limit of detection (3s b /m) was 4.2 μg L -1 of cisplatin. The interfering effect of diverse species was investigated. The developed method was used for the quantification of cisplatin in bio fluids of patients treated with cisplatin, spiked bio fluids and pharmaceutical samples and yielded satisfactory results.

Aim: The association of excision repair cross-complementing 1 mRNA (ERCC-1 mRNA) expression with the outcome has been reported with immunohistochemistry (IHC) using tumor tissue in head and neck cancer. We evaluated ERCC-1 mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) from peripheral blood lymphocytes (PBLs) as bio-predictor of locoregional failure (LRF) to chemoradiation (CRT) for locally advanced laryngeal squamous cell cancer (LALSCC)., Methods: A total of 107 male patients with LALSCC were enrolled in this prospective study. ERCC-1 mRNA expression by PBLs was determined by RT-PCR. Definitive CRT was delivered with 35 mg/m 2 weekly cisplatin. Response Evaluation Criteria in Solid Tumor 1.1 (RECIST 1.1) were used in evaluating treatment response. The primary objective was to assess LRF. The influence of patient characteristics, treatment response, weekly cisplatin cycles, ERCC mRNA expression was determined for LRF, progression-free survival (PFS) and overall survival (OS)., Results: A total of 98 patients completed definitive CRT. The median value of 2-ΔΔCT ERCC-1 mRNA expression was 3.9; based on which it was categorized as low and high. Correlation of ERCC-1 expression with treatment response was insignificant (P- .38). With a median follow-up of 33 months; 2-year LRF, PFS, and OS was 63.3%, 34.7% and 79.4%. The 2-year LRF, PFS and OS for low versus high expression were 53.1% versus 73.5% (P-value = 0.036), 44.9% versus 24.4% (P-value = 0.047) and 81.6% versus 77.2% (P-value = 0.33), respectively. In multivariate analysis, ERCC-1 expression, T-stage, N-stage and tumor subsite are predictive factors for LRF; T-stage and nodal recurrence for OS; stage and treatment response for PFS., Conclusion: LALSCC patient with ERCC-1 mRNA low expression was associated with lower LRF rate, and improved PFS., (© 2019 John Wiley & Sons Australia, Ltd.)

Tumour-associated macrophages (TAMs) are the key components in the tumour microenvironment. TAMs have two major subtypes, M1 and M2. M1 macrophages are tumour inhibitory, while M2 macrophages are tumour promotive. Repolarising TAMs from M2 to M1 is a promising strategy in cancer treatment. M1 and M2 macrophages were generated from murine bone marrow-derived macrophages (BMDMs). We found that chloroquine (CQ), an autophagy inhibitor, was able to repolarise M2 macrophages to the anti-tumour M1 phenotype. The repolarised macrophages demonstrated higher phagocytotic activity towards Hep-2 laryngeal tumour cells and re-sensitised Hep-2 cells to cisplatin (CDDP) treatment in vitro. While CQ did not demonstrate cytotoxicity to Hep-2 cells in vitro, CQ treatment reduced Hep-2 laryngeal tumour growth in vivo and improved CDDP treatment outcomes. It seems that CQ-induced M2-to-M1 macrophage repolarisation played an important role in tumour growth inhibition, and the CQ/CDDP combined therapy might have clinical potential in laryngeal cancer treatment.

In this study, laryngeal tumor cells were killed through the production of excessive reactive oxygen species (ROS) and Ca 2+ influx by cisplatin (CISP). Nevertheless, a resistance was determined against CISP treatment in the tumor cells. We have investigated the stimulating role of curcumin (CURC) on CISP-induced human laryngeal squamous cancer (Hep2) cell death through TRPM2 channel activation, and its protective role against the adverse effects of CISP in normal kidney (MPK) cells. Hep2 and MPK cells were divided into four groups as control group, CURC group (10μM for 24 hrs), CISP group (25 μM for 24 hrs), and CURC + CISP combination group. CISP-induced decrease of cell viability, cell count, glutathione peroxidase and glutathione level in Hep2 cells were further increased by CURC treatment, but the CISP-induced normal MPK cell death was reduced by the treatment. CISP-induced increase of apoptosis, Ca 2+ fluorescence intensity, TRPM2 expression and current densities through the increase of lipid peroxidation, intracellular and mitochondrial oxidative stress were stimulated by CURC treatment. In conclusion, CISP-induced increases in mitochondrial ROS and cell death levels in Hep2 cells were further enhanced through the increase of TRPM2 activation with the effect of CURC treatment. CISP-induced drug resistance in Hep2 cells might be reduced by CURC treatment.

Objectives: To investigate the effect of Procyanidins (OPCs) on the autophagy of laryngeal cancer cell line TU686 and to explore the effect of OPCs on the chemosensitivity of laryngeal cancer cells to DDP in terms of autophagy and apoptosis., Methods: CCK-8 was used to detected the effect of different concentrations of OPC and DDP on TU686 cell viability. Experimental grouping: Both kinds of cells were divided into CON group, DDP group, OPC group and MIX group. Annexin-V-FITC/PI double staining of flow cytometry was used to detect the effect of each experimental group on the apoptosis. Cell immunofluorescence staining was used to detect the formation of autophagy. Western blot was used to detect the expression of autophagy-related and apoptosis-related proteins. Autophagy inhibitors (3-MA) were used to study the effect of autophagy on apoptosis., Results: The results of CCK-8 showed that TU686 cells were inhibited by OPC and DDP in a concentration-dependent manner for 24 hours. LC3-Ⅱ protein staining showed that compared with CON group, DDP group and OPC group, MIX group significantly induced autophagy formation in TU686 cells ( P <0.05). Flow cytometry showed that compared with CON group, apoptosis of TU686 cells was induced in DDP group, OPC group and MIX group. And the effect of MIX on apoptosis was significantly higher than that of OPC and DDP groups ( P <0.05). After pretreatment with 3-MA, the apoptotic effect of OPC group and MIX group on TU686 cells was significantly decreased ( P <0.05). Western blot results showed that the expression of LC3-Ⅱ and Caspase-3 in DDP, OPC and MIX groups was significantly higher than that in CON group ( P <0.05). In MIX group, the expression of LC3-Ⅱ and Caspase-3 also had significant difference ( P <0.05) compared with single drug group. After using 3-MA to inhibit autophagy, the expression of LC3-Ⅱ was significantly decreased ( P <0.05), and the expression of Caspase-3 was decreased along with LC3-Ⅱ, but the decrease of Caspase-3 expression was only significant in OPC and MIX group ( P <0.05)., Conclusions: OPC can induce autophagy in laryngeal carcinoma TU686 cells and promote its apoptosis, which in turn enhances sensitivity of laryngeal cancer cells to cisplatin chemotherapy., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Objective: To study the relationship between transcription factor Snail and the sensitivity of cisplatin on human laryngeal resistant cancer cells. Method: siRNA interference of Snail was transfected by small RNA interference technology. The interference efficiency on mRNA level were detected by RT-qPCR assay; the expression of Snail protein level was assessed by immunofluorescence. The inhibition ratio of different cisplatin concentration (0, 1, 2, 4, 8, 16 μg/ml) was detected by CCK-8 assay; the protein level of Snail, E-cadherin, MDR1were detected by Western blot assay. Result: RT-qPCR assay show the expression of Snail on mRNA level was decreased to (67.85±9.50)% after transfection in Hep-2/CDDP cell( P <0.05). Immunofluorescence show fluorescence intensity of si-Hep-2/CDDP group was reduced both in nucleus and cytoplasm; CCK-8 assay show the inhibitory ratio of transfected group was increased compared to negative control and Hep-2/CDDP group in different cisplatin concentration (0, 1, 2, 4, 8, 16 μg/ml) ( P <0.05). Western blot assay show the protein expression of Snail and MDR1 were down-regulated in transfected Hep-2/CDDP cells (all P <0.05), while epithelial marker E-cadherin was up-regulated in protein level ( P <0.05). Conclusion: Small interference of transcription factor Snail could increase the expression of E-cadherin while decrease the expression of MDR1, and it was confirmed that interference Snail contribute to enhanced cisplatin sensitivity on human laryngeal resistant cancer cells., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Larynx squamous cell carcinoma represents one of the most common head and neck cancers in the world. Herbal drugs are popularly emerging as complementary and alternative therapies in cancer because of their cost effectiveness and minimal side effects. The present study was undertaken to explore the anti-tumor potential of berberine, an isoquinolone present in the extract of Tinospora cordifolia in HEP2 human laryngeal cancer cell line. Besides, it was aimed to investigate whether berberine could enhance the anti-cancer effect of 5-fluorouracil and cisplatin in HEP2. Our data seem to support a role for berberine in decreasing the expression of genes usually seen overexpressed in larynx squamous cell carcinoma and involved in pathways such as those of cell cycle and regulation, differentiation, and epithelial-mesenchymal transition. Moreover, a down regulation of these genes caused by cisplatin or 5-fluorouracil, treatment of election in laryngeal cancers was enhanced by a 4h pre-treatment with berberine.

To investigate the role of Beclin1 in cisplatin-induced apoptosis in laryngeal carcinoma cells Hep-2 and to explore the potential mechanism. We up-regulated Beclin1 expression in Hep-2 cells. The survival rate and apoptotic rate were evaluated by MTT and flow cytometry (FCM). The Beclin1 overexpression group and the control group were treated with cisplatin for 24 hours. The proliferation and cell apoptosis of laryngeal cancer cell lines were evaluated. The mitochondrial membrane potentials were detected by DiOC6(3). Activities of Caspase-8/9/3 and convention of microtubule-associated protein one light chain 3 (LC3) were detected by western blot. The effect of Bcl-2 overexpression on increased cisplatin-sensitivity and autophagy induced by Beclin1 was investigated using Bcl-2 cDNA transfection. Expression of Beclin1 in Hep-2 cells was meaningfully enhanced by transfection, and the proliferation and the apoptosis were not considerably affected. By cisplatin treatment, the Beclin1 overexpression group showed lower survival rate and higher apoptotic rate than the control group (p<0.05). Decrease of mitochondrial membrane potential and increase of activities of Caspase-8, Caspase-9 and Caspase-3 were detected. Beclin1 overexpression increase the convention of LC3, especially after the cisplatin treatment. Overexpression of Bcl-2 decreased the cisplatin-induced apoptosis and inhibited Beclin1-induced autophagy. In conclusion, Beclin1 enhances cisplatin-induced apoptosis in laryngeal carcinoma cells Hep-2 via Bcl-2 modulated autophagy.

Background: Leukocytoclastic vasculitis is typically mediated by deposition of immune complexes and is related to many causes, including medication. To the best of our knowledge, leukocytoclastic vasculitis related to cisplatin has not yet been described in the scientific literature., Case Presentation: We report a rare case of leukocytoclastic vasculitis after the first cycle of high-dose cisplatin chemotherapy in a patient with larynx carcinoma. A 48-year-old Caucasian man with larynx carcinoma received a high-dose of cisplatin monochemotherapy (100 mg/m 2 every 21 days), along with 70 Gy of radiotherapy divided into 35 sessions, as a therapeutic schedule. Twelve days after the first chemotherapy administration and after 8 sessions of radiotherapy (total of 16 Gy), the patient presented with acute onset of palpable purpura in the lower limbs. The patient was hospitalized for 10 days, and during this period, he underwent several examinations to rule out infectious, autoimmune, and neoplastic disorders. A skin biopsy showed leukocytoclastic vasculitis with a positive pattern for IgM and C3, as detected through direct immunofluorescence. Twenty-five days after cisplatin administration, the chemotherapy regimen was changed to carboplatin AUC 5, and the episodes of purpura ceased, reinforcing the hypothesis of an adverse reaction to cisplatin., Conclusions: Cisplatin can induce leukocytoclastic vasculitis and clinicians should be aware of this potential effect for better case management and diagnosis.

Objective: To investigate the mechanism between epithelial-mesenchymal transition (EMT) and cisplatin induced resistant cell subline and the malignant biological characteristics, to explore EMT in human hep-2 laryngeal resistant cells. Method: Using cisplatin-resistant cells (hep-2/CDDP) and non-resistant cells (hep-2) established in our previous study; the invasion and migration biological behaviors were detected by transwell and scratch assay; the expressions of E-cadherin, Zo-1, Snail, Slug, Twist1, Vimentinon in the mRNA level were detected by RT-qPCR and the protein level by Western blot. Result: Transwell and scratch assay show the invasion and migration behaviors were increased in hep-2/CDDP cells ( P <0.05), the epithelial marker E-cadherin and Zo-1 were downregulated in hep-2/CDDP cells (all P <0.05), transcription factor Snail, Slug were upregulated in mRNA and protein level (all P <0.01) while Twist1 had no significant changed in protein level ( P >0.05), the expression of mesenchymal marker Vimentin was also increased in mRNA and protein levels in cisplatin resistant cells ( P <0.01). It was confirmed that the hep-2/CDDP cells possessed EMT phenotypes. Conclusion: The cisplatin resistant laryngeal cancer cells perform higherinvasion and migration biological behaviors,and the mechanisms of increased ability of invasion and migration induced by cisplatin was associated to eEMT, study on signal path related to EMT may overcome cisplatin resistance and reduce invasion and migration behaviors., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Chemoresistance is a major obstacle in chemotherapy of laryngeal carcinoma. Recently, studies indicate that cancer stem cells are responsible for chemotherapy failure. In addition, microRNAs play important roles in tumor initiation, development and multidrug resistance. In the present study, we found that the expression of microRNA-125a was decreased in laryngeal carcinoma tissues and Hep-2 laryngeal cancer stem cells (Hep-2-CSCs). MicroRNA-125a gain-of-function significantly increased the sensitivity of Hep-2-CSCs to cisplatin in vitro and in vivo. Combination with microRNA-125a mimics can decrease the half maximal inhibitory concentration of Hep-2-CSCs to cisplatin. Mechanically, we found that microRNA-125a reverses cisplatin resistance in Hep-2-CSCs by targeting Hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1). Inhibition of HAX-1 by microRNA-125a significantly promotes the cisplatin-induced apoptosis in Hep-2-CSCs through mitochondrial pathway. In addition, multidrug resistance of Hep-2-CSCs to vincristine, etoposide and doxorubicin was greatly improved after the cells were transfected with microRNA-125a mimics. These dates strongly suggested the promotion of microRNA-125a/HAX-1 axis on chemotherapy of laryngeal carcinoma.

Objectives/hypothesis: To evaluate the efficacy of peritumoral hyaluronic acid (HA)-cisplatin therapy in a murine model of laryngeal squamous cell carcinoma and to evaluate its effect on cancer stem cells (CSCs)., Study Design: An orthotopic murine study utilizing University of Michigan squamous cell carcinoma-12 (UMSCC-12) laryngeal cancer cells was conducted in randomized controlled fashion with three treatment arms: saline, systemic cisplatin, and peritumoral HA-cisplatin., Methods: UMSCC-12 laryngeal cancer cells were inoculated into the buccal mucosa of athymic nude mice followed by weekly treatment with saline, systemic cisplatin, or peritumoral HA-cisplatin for 3 weeks. Tumor response and animal weight was monitored and change in CD44 proportion was analyzed ex vivo., Results: HA-cisplatin demonstrated superior antitumor efficacy and greater reduction in CD44 positivity on ex vivo analysis., Conclusions: Peritumoral nanoconjugated HA-cisplatin provides superior antitumor efficacy compared to standard cisplatin therapy in an in vivo laryngeal cancer model. There was also selective targeting of CD44+ cancer cells with HA-cisplatin. This therapeutic strategy could represent the first selective laryngeal CSC-targeted therapy. Further preclinical investigation is warranted to evaluate its role for locally advanced head and neck cancer treatment., Level of Evidence: NA Laryngoscope, 126:E184-E190, 2016., (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

It has been previously reported that cisplatin is a well-known anticancer drug being used against a wide range of malignancies including head and neck, ovarian and non-small cell lung carcinoma, and demonstrated its anticancer activity by reacting with DNA or changing cell structure, immune response, reactive oxygen species level (ROS). In this research we proved that cisplatin induced cell injuries and heme oxygenase-1 (HO-1) expression in laryngeal squamous cell cancer Hep-2 cells through ROS generation. The induction of HO-1 clearly protected Hep-2 cells from cisplatin-induced cell death and ROS reaction, and the inhibitor of HO-1 enhanced the cell death and ROS generation induced by cisplatin. Furthermore, the HO-1 expression induced by cisplatin was strongly inhibited by the knockdown of nuclear factor-erythroid-2-related factor-2 (Nrf-2), and the oxidative damages induced by cisplatin were significantly enhanced. Therefore, it may be concluded that the inhibition of HO-1 or the knockdown of Nrf-2 significantly enhanced cisplatin's anticancer effects on Hep-2 cells. In clinic, with the overexpression of HO-1 in laryngeal squamous cancer tissues, the combination of cisplatin with the inhibitor of HO-1 or Nrf-2 siRNA may act as a new method to the treatment of laryngeal squamous cancer.

Aim: To investigate whether neoadjuvant intra-arterial chemotherapy down-stages the tumors and predicts ultimate outcome in patients with advanced laryngeal and hypopharyngeal cancer., Methods: Fifteen patients with stage III and IV cancers received two cycles of intra-arterial cisplatin (40 mg/m(2)) at days 1 and 15, infused super-selectively into superior thyroid artery. Interim evaluation using RECIST 1.1 and volumetry was done in all patients at day 25. Subsequently, the patients received concurrent chemoradiotherapy or surgery with radiotherapy. Final evaluation was done in 10 patients., Results: At interim evaluation, five patients qualified as responders. Partial response, stable disease and progressive disease rates were 40, 33 and 27%, respectively. At final evaluation, complete response was noted in all patients., Conclusions: At interim evaluation, the response rates were lower compared to intravenous neoadjuvant chemotherapy and response to intra-arterial chemotherapy did not predict the ultimate outcome. Thus neoadjuvant intra-arterial chemotherapy offers no advantage over concurrent chemoradiotherapy., (© 2013 Wiley Publishing Asia Pty Ltd.)

The objectives of this study were to identify specific long noncoding RNAs (lncRNAs) in laryngeal squamous cell carcinoma (LSCC) and to clarify the function of cisplatin and paclitaxel on the confirmed laryngeal cancer lncRNAs. Fifty-four pairs of laryngeal tumor and adjacent normal tissue were collected. Candidate lncRNAs were searched in authorized databases. The significant lncRNAs were identified and confirmed through high-output real-time PCR. Chemotherapy assay evaluated the influences of cisplatin and paclitaxel on the significant lncRNAs. Thirty-seven cancer-related candidate lncRNAs were selected. Three up-expressed and two down-expressed significant lncRNAs were identified and confirmed. The expressions of lncRNA CDKN2B-AS1, HOTAIR and MALAT1 were dramatically reduced with the increasing concentration of cisplatin and paclitaxel and also lengthening of the treatment duration. Cisplatin and paclitaxel have target function on significant lncRNAs in LSCC, which presents novel molecular targets to cure LSCC patients and also leads an orientation for developing new drugs.

Background: Epidermal growth factor receptor (EGFR) overexpression and prognostic value in head and neck squamous cell cancer is the basis for targeting by anti-EGFR antibodies, which increase the efficacy of radiotherapy. In order to evaluate the best therapeutic schedule, the effects of cetuximab (C225) on Hep-2 cell proliferation, alone and in combination with cisplatin, were studied., Methods: Hep-2 cells were treated with cetuximab alone or in combination with cisplatin. After determining cell viability with trypan blue, morphological features of apoptotic degeneration were analysed by fluorescence microscopy with Hoechst 33258 stain., Results: Cetuximab alone mildly inhibited Hep-2 proliferation and showed no pro-apoptotic effects. When administered concomitantly with cisplatin, cetuximab synergistically increased inhibition of proliferation and apoptosis., Conclusion: The antiproliferative activity of cetuximab is consistent with its hypothesised role in inhibiting repopulation. However, the increase in the effects of pro-apoptotic agents induced by cetuximab may be even more relevant to its clinical effectiveness than the inhibition of repopulation.

Laryngeal carcinoma is a common malignant neoplasm occurring in the head and neck, threatening human health. Protein casein kinase 2-alpha (CK2α) has been indicated to participate in the pathogenesis of this cancer; however, the underlying mechanisms still need to be elucidated. In this study, short hairpin RNA (shRNA)-mediated RNA interference (RNAi) technology was utilized to inhibit the CK2α expression in HEp-2 laryngeal carcinoma cells. Results showed that both mRNA and protein expression levels of endogenous CK2α were markedly decreased in HEp-2 cells transfected with CK2α specific shRNA. Transwell assays revealed that stable knockdown of CK2α significantly inhibited the migration and invasion of HEp-2 cells. As compared with cells treated with negative control shRNA, epithelial cadherin (E-cadherin) expression was increased, but snail, slug and vimentin were decreased in cells transfected with CK2α shRNA, indicating that inhibition of CK2α expression may suppress the epithelial-mesenchymal transition (EMT) process of laryngeal carcinoma in vitro. Moreover, suppression of CK2α was found to enhance the apoptosis induced by cisplatin in laryngeal carcinoma cells, probably through inhibition of permeability glycoprotein (P-glycoprotein) and multidrug-resistance protein (MRP1). In conclusion, our study may provide a promising therapeutic strategy for human laryngeal carcinoma by targeting CK2α., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Objective: To investigate the combined effect of thrichosanthin (TCS) and cisplatin on Hep-2 and AMC-HN-8 cell proliferation., Methods: Hep-2 and AMC-HN-8 cells were treated with low (3 µg/ml) or high (10 µg/ml) concentration of cisplatin (10 µg/ml), TCS(5 µg/ml), or the combination TCS (5 µg/ml) and cisplatin (3 µg/ml), the cells no exposure to the drugs as control. Cell proliferation was detected by CCK-8. Toxicity of drugs to cells was evaluated by LDH assay. Flow cytometry was used to detect apoptosis. Western blot was performed to detect the expressions of JNK, phosphor-JNK, p38, phosphor-p38, NF-κB, inhibitor of κB (I-κB), and phospho-I-κB., Results: Compared with TCS (5 µg/ml) or cisplatin (3 µg/ml) alone, the combination of them had more significant inhibitory effect on the proliferation of Hep-2 and AMC-HN-8 cells (P < 0.01), but with no significant increase in cytotoxicity (P > 0.05). Western blot showed the expression of p-JNK/SAPK significantly increased in the cells treated with 5 µg/ml TCS for 48 hours, while the expression of NF-κB and phospho-I-κB increased in the cells treated with 3 µg/ml cisplatin. However in the cells treated with 5 µg/ml TCS combined with 3 µg/ml cisplatin, the expression of p-JNK stayed at a high level and the expressions of NF-κB and phospho-I-κB decreased dramatically compared to the cells treated with 3 µg/ml cisplatin alone., Conclusions: TCS could enhance cisplatin-induced apoptosis in Hep-2 and AMC-HN-8, at least in part, by inhibiting the NF-κB signaling pathway and activating JNK/SAPK signaling pathway and thus strengthening the antitumor effects of cisplatin, which highlights the possibility of combined application of TCS and cisplatin in the treatment of laryngeal carcinoma.

Objective: The purposes of this study were to determine whether autophagy was involved in cisplatin (CDDP) resistance and to investigate the role of the autophagy in the regulation of chemosensitivity to CDDP in laryngeal cancer Hep-2 cells., Methods: A WST-1 assay was performed to determine cell viability and cell proliferation. Autophagy activation and proapoptotic effects were characterized using monodansylcadaverine labeling and Hoechest staining, respectively. Western blot analysis was used to detect the expression of apoptotic and autophagy-related genes. Flow cytometry was used to assess cell apoptosis ratio., Results: Exposure to CDDP induced the aggregation of autophagosomes in the cytoplasms of Hep-2 cells and up-regulated the expression of Beclin 1 and LC3II. However, CDDP treatment could not lead to obvious inhibition of cell proliferation, which implies that the autophagy may protect CDDP-treated cells from undergoing cell death. Meanwhile, the WST-1 assay indicated that knockdown of the autophagic gene Beclin 1 sensitized Hep-2 cells to CDDP. Furthermore, CDDP-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitor 3-methyladenine or small interfering RNA against Beclin 1. For the definite mechanism of Beclin 1-enhancing chemosensitivity to CDDP, we found that Beclin1 augmented CDDP-induced apoptotic signaling via enhancing caspase-9 and caspase-3 activity but not caspase-8., Conclusion: Our results suggest that functional autophagy in response to CDDP may lead to cell survival in Hep-2 cells, whereas defective autophagy may contribute to CDDP-induced apoptosis in Hep-2 cells. Thus, modulators of autophagy may be used beneficially as adjunctive therapeutic agents during the treatment of laryngeal cancer with CDDP therapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Concomitant radiotherapy and superselective arterial infusion of cisplatin for laryngeal cancer has shown excellent therapeutic outcomes. It is expected to be a reasonable treatment option for laryngeal cancer, especially in locally advanced cases.

Purpose: To perform a comprehensive cognitive function (CF) assessment in patients who were relapse free after curative intent radiotherapy (RT) or chemoradiotherapy for squamous cell carcinoma of the head and neck., Methods and Materials: Patients underwent neuropsychological tests to assess their objective CF; completed questionnaires to assess subjective CF, quality of life, and affect; and underwent blood tests to assess hematologic, biochemical, endocrine, and cytokine status. Retrospectively, the dosimetry of incidental radiation to the brain was determined for all patients, and the dose intensity of cisplatin was determined in those who had undergone chemoradiotherapy., Results: A total of 10 patients were enrolled (5 treated with radiotherapy only and 5 with radiotherapy and cisplatin). The mean time from the end of treatment was 20 months (range, 9-41). All patients were able to complete the assessment protocol. Of the 10 patients, 9 had impaired objective CF, with memory the most severely affected. The severity of memory impairment correlated significantly with the radiation dose to the temporal lobes, and impaired dexterity correlated significantly with the radiation dose to the cerebellum, suggesting that these deficits might be treatment related. Patients receiving cisplatin appeared to have poorer objective CF than patients receiving only RT, although this difference did not achieve statistical significance, likely owing to the small sample size. Consistent with the published data, objective CF did not correlate with subjective CF or quality of life. No association was found between objective CF and patients' affect, hematologic, biochemical, endocrine, and cytokine status., Conclusion: Neuropsychological testing is feasible in squamous cell carcinoma of the head-and-neck survivors. The findings were suggestive of treatment-related cognitive dysfunction. These results warrant additional investigation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: The most common chemoradiotherapy regimen is high-dose (100 mg/m(2)) three-weekly cisplatin with concomitant radiotherapy; however, this protocol is associated with acute and late toxicities. Here, we reviewed the dose intensity and toxicity for concomitant weekly cisplatin and radiotherapy in patients with head and neck cancer., Methods: Fifty-three patients with untreated head and neck cancer were enrolled and evaluated at our institution from April 2006 to April 2010. Weekly cisplatin (40 mg/m(2)) was given on weeks 1, 2, 3, 5, 6 and 7 with radiotherapy, which comprised a standard dose of 70 Gy delivered in 35 daily fractions over 7 weeks., Results: Fifty-one patients (96.2%) received the full dose of radiotherapy, while the course was disrupted by adverse events in two. Over the course of the chemotherapy, 31 patients (58.5%) received more than 200 mg/m(2) cisplatin. The toxicity was manageable in all except one patient, who died of sepsis after completing treatment. The 2-year overall survival rate and local progression-free rate for all patients were 93.7% and 88.0%, respectively. The primary site showed a complete response in 52 patients (98.1%) and a partial response in 1 patient (1.9%). The primary disease was well controlled by chemoradiotherapy in 47 patients (88.7%)., Conclusions: Weekly cisplatin could be easier to manage than three-weekly cisplatin, because patients can be monitored more regularly for toxicity allowing the schedule to be altered if required. This regimen appears to be a suitable alternative to three-weekly high-dose cisplatin with concomitant radiotherapy.

CD147, also named extracelluar matrix metalloproteinase inducer (EMMPRIN), is a member of the immunoglobulin family and a glycoprotein enriched on the surface of tumor cells, which promotes invasion, metastasis, growth and survival of malignant cells, and is known to confer resistance to some chemotherapeutic drugs. To determine the possible role of CD147 in the invasive properties of laryngeal carcinoma, we used an RNA interference approach to silence CD147 expression in the Hep2 cell line at high levels of CD147 expression. Our results showed that CD147 expression was significantly impeded at both mRNA and protein levels, which resulted in a decrease of the Hep2 invasion activity in vitro and tumorigenicity in nude mice. The suppression of CD147 expression also sensitized cells to cisplatin. Our current results indicated that CD147 was a laryngeal carcinoma-related gene and CD147 might be a potential target for therapeutic anti-cancer drugs.

Acquired resistance to cisplatin represents a major obstacle to an efficient chemotherapy. We found downregulation of RhoB expression in cisplatin-resistant tumor cell lines from different origin. In cisplatin-resistant laryngeal carcinoma subline overexpression of farnesylated or geranylgeranylated RhoB increased cisplatin-induced cell death, while silencing of RhoB expression diminished sensitivity of parental HEp-2 cells via decreased cellular accumulation of cisplatin. However, since RhoB silencing in additional tumor cell lines did not alter their sensitivity to cisplatin, we can assume that RhoB downregulation does not provide general protective role in cell response to cisplatin. Nevertheless, gene therapy involving restoration of RhoB expression might improve the efficiency of cisplatin treatment, especially in patients with laryngeal carcinoma that acquired resistance to this chemotherapeutic drug., (2010 Elsevier Ireland Ltd. All rights reserved.)

Adverse events and therapeutic effects were analyzed in patients with pharyngeal or advanced laryngeal squamous cell carcinomas(SCCs)receiving concurrent chemoradiotherapy (CCRT) with S-1 or weekly CDDP between 2004 and 2007. Low-dose CDDP (25 mg/m2) was administered once a week and S-1 (65 mg/m2) was administered for 3 weeks with one-week rest during conventional radiation with 2 Gy/fraction. Both of the two CCRT regimens showed little toxicity with grade 4 toxicities in less than 5%of the patients. However, CCRT with S-1 more frequently induced grade 3 and 4 oral mucositis than CCRT with CDDP. As a result, the completion rate of CCRT with S-1 was lower than that of CCRT with CDDP. The two regimens achieved a similar complete response rate of the primary sites, local control rate(LCR)and larynx preservation rate; the LCR for T1 and 2 disease was more than 70%. However, the LCR for T3 or 4 disease by the two regimens was less than 50%. CCRT with S-1 showed significantly higher LCR in patients with poorly or undifferentiated SCCs than those with well or moderately-differentiated SCCs. It is suggested that the two CCRT regimens are useful treatment modalities for patients with locally(primary site)non-advanced pharyngeal or laryngeal SCCs, and that CCRT with S-1 is highly sensitive to poorly or undifferentiated SCCs. In order to achieve local control and larynx preservation, more intensive CCRT might be necessary for patients with locally(primary site)advanced pharyngeal or laryngeal SCCs.

Purpose: To determine the influence of concomitant radiochemotherapy with cisplatin on parotid gland tissue complication probability., Methods and Materials: Patients treated with either radiotherapy (n = 61) or concomitant radiochemotherapy with cisplatin (n = 36) for head-and-neck cancer were prospectively evaluated. The dose and volume distributions of the parotid glands were noted in dose-volume histograms. Stimulated salivary flow rates were measured before, during the 2(nd) and 6(th) weeks and at 4 weeks and 6 months after the treatment. The data were fit using the normal tissue complication probability model of Lyman. Complication was defined as a reduction of the salivary flow rate to less than 25% of the pretreatment flow rate., Results: The normal tissue complication probability model parameter TD(50) (the dose leading to a complication probability of 50%) was found to be 32.2 Gy at 4 weeks and 32.1 Gy at 6 months for concomitant radiochemotherapy and 41.1 Gy at 4 weeks and 39.6 Gy at 6 months for radiotherapy. The tolerated dose for concomitant radiochemotherapy was at least 7 to 8 Gy lower than for radiotherapy alone at TD(50)., Conclusions: In this study, the concomitant radiochemotherapy tended to cause a higher probability of parotid gland tissue damage. Advanced radiotherapy planning approaches such as intensity-modulated radiotherapy may be particularly important for parotid sparing in radiochemotherapy because of cisplatin-related increased radiosensitivity of glands.

A prospective multicenter phase-II trial (12 centers) was performed by the German larynx organ preservation group (DeLOS) to evaluate the effect of induction chemotherapy (ICHT) with paclitaxel/cisplatin (TP), followed by accelerated-hyperfractionated (concomitant boost) radiotherapy (RT) in responders. The trial was focused on larynx preservation, tumor control, survival, salvage surgery and late toxicity in patients with advanced larynx/hypopharynx carcinoma eligible for total laryngectomy (LE). Seventy-one patients (40 larynx, 87.5% St. III, IV; 31 hypopharynx, 93.4% St. III, IV) were enrolled into the study and treated with ICHT (200 mg/m(2) paclitaxel, 100 mg/m(2) cisplatin; day 1, 22) according to the DeLOS protocol. Patients with complete or partial tumor response proceeded to RT (69.9 Gy in 5.5 weeks). Non-responders received a LE followed by postoperative RT (56-70 Gy in 5.5-7 weeks). The response rate to ICHT for larynx cancer was 69.6% (7.1% complete, 62.5% partial response) and for hypopharyngeal cancer was 84.3% (6.9% complete, 77.4% partial response). Overall survival after 36 months was 60.3% (95% CI, 48.4-72.2%), after 42 months was 56.5% (95% CI, 44.2-68.8%). Laryngectomy-free survival was as follows: after 36 months, 43.0% (95% CI, 30.9-55.0%); after 42 months, 41.3% (95% CI, 29.3-53.3%). Both parameters did not show different outcomes after distinguishing larynx from hypopharynx. LE was indicated in 15 non-responders after ICHT. Five of the 15 non-responders refused the laryngectomy. Two of the five received RT instead and had no evidence of disease 42 months after RT. Late toxicity (dysphagia III, IV LENT SOMA score in laryngectomy-free survivors: after 6 months, 1.8%; 12 months, 11.4%; 18 months, 14.5%; 24 months, 8.1%; 36 months, 16%) and salvage surgery (4 pharyngocutaneous fistulas in 27 operations) were tolerable. In a large portion of patients eligible for LE, the larynx could be preserved with satisfying functional outcome. Good responders after ICHT had also a good general outcome with relatively rare severe late toxicities. Due to a slight increase of relevant late dysphagia, functional outcome regarding swallowing and tracheotomy free breathing should be more focused in future larynx organ preservation trials.

Objectives: We had previously treated patients with advanced stage laryngeal cancer by laryngectomy with or without post-operative radiotherapy. In order to improve such patients' quality of life, we sought to preserve the larynx by selective (intra-arterial), rapid infusion chemotherapy combined with radiotherapy., Methods: Chemotherapy was administered intra-arterially in the angiography suite via transfemoral catheterisation of the superior thyroid artery. Patients received up to four once-weekly infusions of cisplatin (75 mg/patient) with simultaneous intravenous administration of sodium thiosulphate, a neutralising agent. Patients also received external radiation simultaneously at a dose of 1.8 or 2.0 Gy per fraction, once daily for five days a week for 7 weeks., Results: Intra-arterial infusion chemo-radiotherapy was performed in eight patients with advanced laryngeal carcinoma (four glottic, three supraglottic and one subglottic type carcinoma). A complete response was achieved at the primary site and at lymph node metastases in all eight patients. Overall toxic side effects were modest. No catheter-related thrombo-embolic complications were observed during any of the chemotherapy sessions., Conclusions: Selective (intra-arterial), rapid infusion chemo-radiotherapy may enable laryngeal preservation in patients with advanced laryngeal carcinoma.

The cytotoxic effects and mechanism of action of cisplatin and the mTOR inhibitor rapamycin on Hep-2 laryngeal cancer cells were investigated. Hep-2 cells were cultured in the presence of different concentrations of rapamycin, cisplatin, or the two combined. Inhibition of cell growth, apoptosis, and AKT, mTOR, S6K, and ERCC1 protein levels were assessed. All combinations of rapamycin and cisplatin resulted in synergistic inhibition of cell growth (as indicated by q values determined using Jin's formula > 1.15). Rapamycin inhibited Hep-2 cell growth, induced G1 arrest, and when combined with cisplatin, enhanced apoptosis. p-mTOR and S6K expressions were significantly downregulated by rapamycin. ERCC1 expression was significantly upregulated with cisplatin treatment. Combined cisplatin and rapamycin treatment resulted in significant downregulated p-mTOR and S6K expression, but no change in ERCC1 expression. Rapamycin and cisplatin act in a synergistic manner, increasing the cytotoxic effect on Hep-2 cells. Rapamycin may facilitate increased Hep-2 cell apoptosis with cisplatin via inhibiting downstream expression of proteins in the AKT-mTOR signaling pathway.

Acquired resistance to cisplatin represents a major obstacle to successful chemotherapy. We have developed cisplatin-resistant CA3(ST) and CK2 cells, which exhibited altered formation of cell-cell junctions compared to their parental cisplatin-sensitive human laryngeal carcinoma HEp-2 cells. Although cell-cell adhesion can induce antiapoptotic signaling, there is contradictory evidence considering the significance of cadherin-catenin complex in cellular response to cisplatin. Therefore, we analyzed junctional proteins in this model of cisplatin resistance. In both cisplatin-resistant sublines plakoglobin expression was decreased, while beta-catenin expression was increased, at cell-cell junctions. Although cisplatin-resistant cells showed decreased plakoglobin mRNA, they retained equal expression of beta-catenin mRNA as parental cells. Immunoprecipitation of cadherin-catenin complex established that upregulation of beta-catenin results from its stabilization through interaction with N-cadherin. Furthermore, beta-catenin upregulation was closely associated with cisplatin exposure, since cisplatin-resistant HeLa subline also had increased beta-catenin, while vincristine-resistant HEp-2 subline did not upregulate beta-catenin. However, single cisplatin treatment of HEp-2 cells did not induce beta-catenin upregulation, nor plakoglobin mRNA downregulation, suggesting that the alteration in catenin ratio is a late event, which requires repeated cisplatin exposure. Finally, we overexpressed plakoglobin in CA3(ST) cells and selected several clones that established the pattern of plakoglobin/beta-catenin expression found in HEp-2 cells. However, none of the clones restored sensitivity to cisplatin. Thus, it appears that beta-catenin and plakoglobin are not involved in the resistance development, implying that the observed alterations are an outcome of a slowly generating process, which is presumably a secondary event of vital cellular response triggered by cisplatin toxicity.

We report a case of advanced small cell carcinoma in the larynx, which was treated with Irinotecan hydrochloride (CPT-11) chemotherapy. The patient was free of disease for 4 years after treatment. Several chemotherapeutic agents for small cell carcinoma have been proposed; however, median survival time has been miserable, especially in advanced cases. For the cure of the aggressive lethal behavior of this disease, chemotherapy with CPT-11 might be effective to improve median survival of patients with small cell carcinomas of the larynx.

We evaluated whether low-dose chemotherapy could improve effects of radiation therapy for glottic carcinoma with different prognostic factor based on the UICC 6th edition. Fifty-one patients with T2N0 glottic carcinoma classified by the UICC 5th edition underwent chemoradiation therapy with low-dose CDDP (4 mg/m(2)) and oral UFT (450 mg of tegafur) continuing for four weeks (CRT group). The historical control consisted of 49 patients treated with radiation therapy alone (RT group). Forty-six tumors with the adjacent sign, i.e. tumors located adjacent to the thyroid cartilage on radiological examinations, were classified as T3 according to the 6(th) edition. The 5-year local control and laryngeal preservation rates of the T2 (n=54) vs. T3 (n=46) lesions were 87% vs. 50% (p<0.0001) and 94% vs. 61% (p<0.0001), respectively. Among the T3 lesions, CRT (n=24) yielded significantly higher laryngeal preservation rates than did RT alone (n=22) (83% vs. 40%, p=0.0063), and the local control rates were higher in the CRT- than the RT group (62% vs. 36%, p=0.0882). While such benefits of CRT were not observed in patients with T2 lesions.

To investigate the response of tumour growth to cisplatin treatment, in relation to p53 mutation and cyclin D1 dysregulation on DNA and protein level, biopsies from seven xenografted human squamous cell carcinomas from the head and neck were analysed with immunohistochemistry for p53 expression and cyclin D1 expression. Polymerase chain reaction-singlestranded conformation polymorphism was used to determine p53 mutations. Fluorescence in situ hybridization was performed to analyse cyclin D1 amplification. The mice were injected i.p. with NaCl (controls) or cisplatin. After injection the tumour volume were measured. The inhibition of tumour growth by cisplatin was defined as the area under the growth curves, and compared with the growth curves of the tumours in the control group. Xenografts with p53 mutation showed significantly higher resistance to cisplatin (p < 0.001) and also tumours with cyclin D1 amplification showed significantly higher resistance (p < 0.001).

A 55-year-old man with cervical radiculopathy (C5-C8) was referred to us following intra-arterial infusion of cisplatin (CDDP) because of a recurrent neck mass of laryngeal cancer. Three hours after the CDDP infusion, he had noticed general weakness of the left upper extremity and hypoaesthesia of the lateral side of the upper and lower arm. The next day he was diagnosed with left cervical radiculopathy of C5 to C8, which improved gradually and had resolved completely six months after the infusion. Even with proper positioning of the infusion catheter to minimize potential complications, for anatomical reasons there are always some risks of neural injury with intra-arterial infusion from branches of the subclavian artery. This procedure should be carefully indicated in the case of a large neck tumour that is perfused from the major branches of the subclavian artery.

Since its discovery by Rosenberg in 1965, cisplatin and its derivatives have appeared as the most important chemotherapeutic agents, particularly for their radiosensitizing properties and their clinical use with radiation. In spite of numerous preclinical and clinical studies, optimal schedules of platin and radiotherapy combination have to be defined. The first part of this overview will describe biological mechanisms of interaction between radiation therapy and platinum derivatives. The second part will report the major clinical impact of their association.

Objective: To investigate the prevalence of hearing loss after concomitant radiochemotherapy in patients enrolled in a larynx preservation protocol., Design: Prospective study., Setting: Consecutive patients treated in a tertiary cancer center hospital between 2001 and 2002., Patients: Eligible subjects included patients prospectively enrolled in an organ preservation protocol based on concomitant radiotherapy and chemotherapy (cisplatin and paclitaxel)., Main Outcome Measures: Descriptive analysis of the results of audiologic evaluations, including pure-tone audiometry and immitance audiometry, which were performed prior to and 8 months after treatment. Change in hearing sensitivity was computed relative to baseline measures. Criteria to indicate hearing decrease after the treatment were defined as either a 20-dB decrease at any single test frequency or a 10-dB decrease at any 2 adjacent test frequencies., Results: A total of 11 patients were analyzed. Four patients (36%) had hearing loss after the treatment., Conclusion: Our results suggest that the prevalence of hearing loss after radiochemotherapy in larynx preservation protocols is high (36%); however, it was usually mild and asymptomatic.

Cisplatin (CDDP) is among the most widely used and most effective chemotherapeutic agent for many types of human cancer. Because killing cancer cells by chemotherapy is principally executed by apoptosis, a defective apoptotic program might acquire drug resistance. Flow cytometric Annexin V assay demonstrated that HEp-2 cells (human laryngeal cancer) were persistently resistant to CDDP as compared to HeLa cells (human uterine cervical cancer), despite the same histological type and wild-type p53 status. CDDP treatment caused steady induction of p53 protein in both cancer cell types, although it was more dramatic in CDDP-resistant HEp-2 cells, which was correlated well with p53 Ser15 phosphorylation, but not with the expression level of HPV type 18 E6 oncoprotein in these cells. Importantly, CDDP differently activated caspase cascades between HEp-2 and HeLa cells. CDDP activated the caspase-8 pathway through TNFR superfamily receptors such as Fas, but not caspase-9 in HeLa cells. On the other hand, the caspase-9 pathway was significantly activated in HEp-2 cells, although the activation of caspase-8 by CDDP was deficient. This different response to CDDP in caspase-8 activation was not related with the expression level of either Fas or FasL in these cells. We concluded from these results that loss of the caspase-8 activation pathway in HEp-2 cells was a possible mechanism for its resistance to CDDP-induced apoptosis. The caspase-8 pathway might play an important role in CDDP-induced apoptosis in HPV-positive human squamous cell carcinomas.

This paper describes the effect of dipyridamole (DIP) on the cytotoxicity of cisplatin in HEp-2 human larynx cancer cells in vitro and the nature of the interaction between cisplatin and dipyridamole. Cytotoxic assays were performed to obtain the IC50 for cisplatin. The cells were treated with 0, 20, 40, 80, 120 or 200 microM cisplatin, with or without a single concentration of DIP and incubated for 60 min at 37 masculine C and 5% CO2 for 3 days and then counted with a hemocytometer. The accumulation of cisplatin in the cells was measured by atomic absorption and fluorescence was used to determine the membrane binding constant of DIP. In the presence of 10, 20 and 30 microM DIP, the IC50 of cisplatin was reduced by 25, 60 and 82% in HEp-2 cells. Combination index analysis revealed that cisplatin and DIP interact synergistically. In larynx cancer cells, the accumulation of cisplatin increased by 13, 27 and 65% as the DIP concentration was increased from 10 to 20 and 30 microM, respectively. The binding constant of DIP to the cell membrane was estimated to be 0.36 +/- 0.12 mg/ml(-1) (N = 2) by fluorescence and cisplatin did not suppress DIP fluorescence. These results suggest that DIP significantly enhances cisplatin cytotoxicity in HEp-2 cells by increasing cisplatin accumulation, probably by altering the cell membrane as suggested by its binding constant. The results obtained reinforce the importance of combination therapy to reduce the doses of chemotherapeutic drugs and therefore the side effects of chemotherapy.

Summary: ABSTRACT The purpose of this study was to achieve locoregional control of locally advanced laryngeal carcinoma, survival, and organ preservation using split hyperfractionated accelerated radiation therapy and cisplatin concomitantly. This study was a phase II trial of chemoradiotherapy with split hyperfractionated accelerated radiation therapy, 1.6 Gy per fraction given twice per day to a total dose of 64 to 67.2 Gy for a total of 6 weeks with a 2-week gap, and cisplatin 20 mg/m2, days 1 to 5, in continuous perfusion, concomitantly. Seventy-three patients were treated (stage IV, 64%). At a median follow-up of 55 months for living patients, median survival was 44 months, and 5-year overall survival and disease-free survival were 42% and 39%, respectively. Toxicities included mucositis (grade III, 40%; grade IV, 28%), epithelitis (grade III, 28%). Of the 73 patients, 32 (44%) have continued with their larynx free of disease. Split hyperfractionated accelerated radiation therapy and concomitant cisplatin has been demonstrated to be an active treatment for locally advanced laryngeal carcinomas, but more active combinations of chemotherapy and radiotherapy, without increase of toxicity, are necessary to increase the rate of locoregional control, organ preservation, and survival.

Background: Induction chemotherapy with cisplatin plus fluorouracil followed by radiotherapy is the standard alternative to total laryngectomy for patients with locally advanced laryngeal cancer. The value of adding chemotherapy to radiotherapy and the optimal timing of chemotherapy are unknown., Methods: We randomly assigned patients with locally advanced cancer of the larynx to one of three treatments: induction cisplatin plus fluorouracil followed by radiotherapy, radiotherapy with concurrent administration of cisplatin, or radiotherapy alone. The primary end point was preservation of the larynx., Results: A total of 547 patients were randomly assigned to one of the three study groups. The median follow-up period was 3.8 years. At two years, the proportion of patients who had an intact larynx after radiotherapy with concurrent cisplatin (88 percent) differed significantly from the proportions in the groups given induction chemotherapy followed by radiotherapy (75 percent, P=0.005) or radiotherapy alone (70 percent, P<0.001). The rate of locoregional control was also significantly better with radiotherapy and concurrent cisplatin (78 percent, vs. 61 percent with induction cisplatin plus fluorouracil followed by radiotherapy and 56 percent with radiotherapy alone). Both of the chemotherapy-based regimens suppressed distant metastases and resulted in better disease-free survival than radiotherapy alone. However, overall survival rates were similar in all three groups. The rate of high-grade toxic effects was greater with the chemotherapy-based regimens (81 percent with induction cisplatin plus fluorouracil followed by radiotherapy and 82 percent with radiotherapy with concurrent cisplatin, vs. 61 percent with radiotherapy alone). The mucosal toxicity of concurrent radiotherapy and cisplatin was nearly twice as frequent as the mucosal toxicity of the other two treatments during radiotherapy., Conclusions: In patients with laryngeal cancer, radiotherapy with concurrent administration of cisplatin is superior to induction chemotherapy followed by radiotherapy or radiotherapy alone for laryngeal preservation and locoregional control., (Copyright 2003 Massachusetts Medical Society)

Background: Simultaneous radiochemotherapy (sRCT) is the treatment of first choice in locally advanced head and neck cancers. We have tested a very aggressive combination protocol with cisplatin and escalated paclitaxel in combination with accelerated hyperfractionated radiotherapy to assess the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), overall toxicity, and response rate., Patients and Methods: The trial recruited 24 patients (21 males, three females, mean age 57 years) treated at our department from 1998 through 2001. Irradiation was administered in daily doses of 2 Gy up to 30 Gy followed by 1.4 Gy twice daily up to 70.6 Gy to the primary tumor and involved nodes and 51 Gy to the clinically negative regional nodes. The chemotherapy schedule included cisplatin in a fixed dose of 20 mg/m(2) on days 1-5 and 29-33 and paclitaxel at increasing dose levels of 20, 25, 30 mg/m(2) twice weekly over the whole treatment time. Patients were recruited in cohorts of three to six, and the MTD was reached if two out of six patients in one cohort developed DLT. DLT was defined as any grade 4 toxicity or any grade 3 toxicity requiring treatment interruption or unplanned hospitalization or any grade 3 neurotoxicity. We recruited mainly patients with large tumors for this protocol; all patients were stage IV, and the mean tumor volume (primary + metastases) amounted to 72 +/- 61 cm(3). The mean follow-up was 30 months (range 4-39 months)., Results: One early death (peritonitis and sepsis at day 10) occurred, and 23 patients were evaluable for acute toxicity and response. The MTD of paclitaxel was reached at the third dose level (30 mg/m(2) paclitaxel twice weekly). The DLT was severe mucositis grade 3 (n = 1) and skin erythema grade 4 (n = 2). After determining the MTD, another 14 patients were treated at the recommended dose level of paclitaxel with 25 mg/m(2) twice weekly. In summary, 13/23 patients (57%) developed grade 3 and 10/23 (43%) grade 2 mucositis. Two patients (9%) had grade 4, five (22%) grade 3, and 16 (69%) grade 2 dermatitis. One patient died at day 30 of neutropenic infection. In one patient, a grade 2 nephrotoxicity appeared requiring cessation of cisplatin chemotherapy. 18/23 patients (78%) required blood transfusion (1-3 units) and 16/23 (70%) i.v. antibiotics. 14 patients (61%) achieved a complete and nine (39%) a partial remission, yielding an overall response rate of 100%. In summary, six patients died of local tumor progression (n = 2), distant metastases (n = 2), or therapy-related complications (n = 2) during follow-up. The 3-year overall survival was 71%. Tumor volume was not a risk factor for failure in this protocol (mean tumor volume in relapse-free vs. progressive patients 71 +/- 65 cm(3) vs. 64 +/- 38 cm(3)). All patients have, so far, developed only slight late effects (fibrosis, lymphedema) with no grade 3-4 late sequelae., Conclusions: This very aggressive sRCT protocol yielded excellent response and survival figures but was associated with a very high rate of acute toxicity (8% therapy-related deaths). A maximal supportive treatment is therefore required.

Based on an inception cohort of 35 patients with T1-3N0M0 squamous cell carcinoma of the true vocal cord who had a complete clinical response after a platinum-based induction chemotherapy regimen and a minimum of 3 years of follow-up, the current retrospective study documented the long-term results and consequences of local recurrence following the use of a platinum-based chemotherapy-alone regimen for cure. During the years 1985 to 1996, 231 patients with invasive squamous cell carcinoma of the true vocal cord classified as T1-3N0M0 were managed at our department with a platinum-based induction chemotherapy regimen. A complete clinical response was achieved in 77 patients. Thirty-five of the 77 patients with complete clinical response were managed at our institution with a platinum-based chemotherapy-alone regimen. The statistical analysis of data on survival, local control, nodal control, distant metastasis, and metachronous second primary tumor incidence was based on the Kaplan-Meier product limit method. Univariate analysis was performed for potential statistical relation between local recurrence and various variables. The 3- and 5-year actuarial survival estimates were 91.4% and 88.6%, respectively. Overall, the causes of death were intercurrent disease in 6 patients and metachronous second primary tumor in 4 patients. The 3- and 5-year actuarial local control estimate was 64.8%. No significant statistical relation could be demonstrated between the incidence for local recurrence and the variables under analysis. Salvage treatment in patients with local recurrence yielded a 100% local control rate and laryngeal preservation rate. The 3- and 5-year actuarial lymph node control estimate was 97.1%. The 3- and 5-year actuarial estimate for patients with distant metastasis was 0%. The 5- and 10-year actuarial estimates for patients with metachronous second primary tumor were 9.7% and 28.1%, respectively. Although local recurrence was noted in almost a third of patients with complete clinical response who were managed with a platinum-based chemotherapy-alone regimen, it did not appear to be detrimental, as none of the patients who had local recurrence ultimately died from their disease or lost their larynx.