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A recent study showned that complementary medicine is gradually gaining wide acceptance. In the present study, the herbal mixture extract (H3) composed of 3 oriental herbal plants was investigated for anticancer activity in vitro and in vivo. H3 inhibited PANC1 cell growth by promoting G0/G1 arrest (11% increase) and apoptotic cell death (9% increase). H3 also suppressed stem cell-like side population cells (4% decrease) and migration activity (24% decrease). In contrast, gemcitabine decreased side population cells and migration activity by 3 and 11%, respectively. These effects of H3 and gemcitabine were further studied by examining the expression of apoptosis-associated genes (CXCR4, JAK2 and XIAP) and stem cell-associated genes (ABCG2, POU5F1 and SOX2). We also found that H3 suppressed tumor growth by 46% in a PANC1‑xenograft model, while gemcitabine caused a 36% decrease. The antitumor effects of H3 were confirmed by western blot analysis for COX-2 and cytochrome c expression. Furthermore, necrotic cell death and erythrocyte-containing cavities were detected in tumor tissue by hematoxylin and eosin (H&E) staining. Notably, the combinatorial therapy (H3 and gemcitabine) increased tumor growth compared to that in the control. In conclusion, the present study shows that H3 has promise as a therapeutic agent against pancreatic cancer and its cancer stem cells.

Development of an efficient treatment for triple-negative breast cancer is an urgent issues. Compounds from plant extracts are a potential source of novel cancer treatment. Isolinderalactone, a kind of sesquiterpenoids compound, was purified from the root of Lindera strychnifolia and Neolitsea daibuensis and shows anti-inflammatory and anticancer capacity. In the present study, isolinderalactone induced apoptosis in MDA-MB-231 cells which is a kind of triple-negative breast cancer cell line through induction of an intrinsic mitochondria-mediated and caspase-independent cell death. Treatment of isolinderalactone increased the protein level of the suppressor of cytokine signaling 3 (SCOS3), decreased phosphorylation of the signal transducer and activator of transcription 3 (STAT3), and suppressed expression of the down-stream genes of the X-linked inhibitor of apoptosis protein in MDA-MB-231 cells. Our results further showed that the level of SOCS3 expression was induced by isolinderalactone due to inhibiting the microRNA hsa-miR-30c-5p (miR-30c) expression. In addition, intraperitoneal injection of isolinderalactone induced apoptosis in a xenograft breast tumor while it did not significantly affect the histology of liver, kidney and lung of the treated mice. In conclusion, isolinderalactone induces apoptosis in MDA-MB‑231 cells and suppresses STAT3 signaling pathway through regulation of SOCS3 and miR-30c. It may become a novel treatment for triple-negative breast cancer in the future.

Epithelial ovarian cancer (EOC) accounts for 90% of all ovarian cancer, which is the third most common gynaecological malignancy worldwide. Dysregulation of miRNAs is involved in the development of different types of EOC. The present study was designed to investigate the role of abnormal expression of miR-215 in the development of EOC and to elucidate the possible molecular mechanisms. mRNA expression of miR-215 was significantly decreased in EOC tissues and cell lines. Upregulation of miR-215 inhibited cell proliferation, promoted apoptosis and increased sensitivity to chemotherapy drugs in EOC cells. In contrast, downregulation of miR-215 increased cell proliferation, inhibited apoptosis and decreased sensitivity to chemotherapy drugs in EOC cells. In addition, the X-chromosome-linked inhibitor of apoptosis (XIAP) expression was significantly increased in EOC tissues and cell lines. Downregulation of XIAP inhibited cell proliferation, promoted apoptosis and increased sensitivity to chemotherapy drugs in EOC cells. Upregulation of miR-215 notably inhibited the expression of XIAP. Moreover, overexpression of XIAP significantly inhibited miR-215-exerted decrease of proliferation, increase of apoptosis and increase of sensitivity to chemotherapy drugs. In conclusion, we identified miR-215 as a potential tumor suppressor in patients with EOC downregulating expression of the oncogenic regulator XIAP. The data demonstrate that miR-215/XIAP pathway may serve as novel therapeutic targets and prognostic markers in patients with EOC.

X-linked inhibitor of apoptosis protein (XIAP) has been shown to be highly expressed in lung cancer, but not in normal lung tissue, which makes it an attractive target for lung cancer treatment. Celecoxib (CXB), a cyclooxygenase-2 inhibitor, is in wide clinical use for the treatment and prevention of non-small cell lung cancer (NSCLC). Therefore, in our study, we combined short hairpin RNA (shRNA) targeted to XIAP (XIAP-shRNA) with CXB and tested the effects of this combination on lung cancer cells to identify more effective therapeutics against lung cancer. An XIAP-shRNA plasmid was constructed and transfected into the A549 NSCLC cell line. The cells were then treated with CXB and XIAP-shRNA alone or in combination for indicated time periods, and the treatments were assessed for their effects on cell proliferation, apoptosis, migration, invasion and receptor signaling using the MTT, TUNEL, wound healing and Matrigel invasion assays and western blotting, respectively. In addition, an NSCLC xenograft model was prepared to observe tumor growth. It was found that both CXB and XIAP‑shRNA significantly inhibited cell proliferation, migration and invasion, and induced cell apoptosis in vitro, as well as suppressed tumor growth in vivo. Moreover, the combination of the agents significantly enhanced these effects compared to the single agent treatments. We also found that the combination treatment significantly suppressed constitutive phosphorylation of PI3K and AKT, which may contribute to the inhibition of tumor growth. These findings suggest that the combination of XIAP‑shRNA and CXB is a promising drug candidate for the treatment of NSCLC.

X-chromosome-linked inhibitor of apoptosis protein (XIAP) is an important member of the inhibitors of apoptosis (IAP) family. It has been shown that XIAP promotes the invasion, metastasis, growth and survival of malignant cells, and confers resistance to some chemotherapeutic drugs in various types of cancer. However, little is known regarding its detailed role in osteosarcoma (OS). In the present study, we first investigated the expression of XIAP in OS tissues, and an increased expression of XIAP in OS tissues compared to adjacent non-tumor tissue was identified. Additionally, its expression level correlated with key pathological characteristics including clinical stage, tumor size and metastasis. Subsequently, small interfering RNA (siRNA) was used to block XIAP expression to evaluate the effect of XIAP siRNA on cell proliferation, colony formation, cell cycle, apoptosis, tumorigenicity, and the combined effects of doxorubicin or cisplatin in OS cell lines (MG63 cells). Downregulation of XIAP expression using the RNA silencing approach efficiently decreased cell proliferation and colony formation, and induced cell apoptosis and cell cycle in the G0/G1 stage. In addition, this downregulation inhibited tumor growth in a nude murine model. The resuls also showed that treatment with XIAP-shRNA in combination with doxorubicin or cisplatin enhanced chemosensitivity. These results suggested that XIAP may aid the diagnosis of OS and may be an effective strategy for gene therapy of this disease.

MicroRNA (miR)-216a expression is significantly downregulated in human pancreatic cancer, however, the underlying mechanism remains unknown. In the present study, we aimed to identify and characterize the direct target gene and potential function of miR-216a in pancreatic cancer cells. Bioinformatics analysis and dual-luciferase reporter gene assay showed that Janus kinase 2 (JAK2) was a direct target gene of miR-216a. Quantitative polymerase chain reaction and western blot analysis demonstrated that miR-216a decreased the mRNA and protein levels of JAK2 in pancreatic cancer cells. Phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was also downregulated by miR-216a, whereas the anti-miR-216a treatment had an opposite effect. Treatment of pancreatic cancer cells with miR-216a significantly inhibited cell growth and promoted cell apoptosis. In addition, the downstream genes of JAK2/STAT3, survivin and X-linked inhibitor of apoptosis protein, which are anti‑apoptotic genes, were also decreased by miR-216a. Moreover, miR-216a overexpression markedly inhibited the JAK2/STAT3 signaling pathway and xenograft tumor growth in vivo. Compared with miR-216a treatment, anti-miR-216a treatment exhibited opposite effects throughout the entire experiment, confirming the inhibitory effect of miR-216a on pancreatic cancer by regulating the JAK2/STAT3 signaling pathway. The results provided evidence that miR-216a targeting JAK2 negatively regulated the development of pancreatic cancer cells and may be used to develop a miRNA-based therapeutic strategy against pancreatic cancer.

Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolarix kaempferi Gordon (Pinaceae). Recent studies have reported that PAB exhibits cytotoxic effects in several cancer cell lines. In the present study, we assessed its antitumor activity and molecular mechanisms in HO-8910 and A2780 ovarian cancer cells in vitro. We found that PAB reduced cell viability and induced apoptosis in a dose- and time-dependent manner in HO-8910 and A2780 human ovarian cancer cells. The induction of apoptosis was also accompanied by the regulation of Bcl-2 and XIAP family proteins, cytochrome c and Apaf-1. Moreover, we observed that PAB treatment resulted in the activation of caspase-3 and -9, which may partly explain the anticancer activity of PAB. Collectively, the present study for the first time suggests that PAB enhances apoptosis of HO-8910 and A2780 cells through regulation of Bcl-2 and IAP family proteins. Moreover, the triggering of caspase-3 and -9 activation mediated apoptotic induction. Our results suggest that PAB may be a new therapeutic option for the treatment of ovarian cancers.

Although cisplatin (CDDP) is a key drug in the treatment of esophageal squamous cell carcinoma (ESCC), acquired chemoresistance remains a major problem. Combination therapy may represent one strategy to overcome this resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition by small molecules, either alone or in combination, has shown promise in the treatment of solid malignancies. In the present study, we evaluated the synergistic effects of combining CDDP with the HSP90 inhibitor 17-N-allylamino-17-demethoxy geldanamycin (17-AAG) on two CDDP-resistant human esophageal squamous cancer cell lines, KYSE30 and KYSE150. The results obtained demonstrated the synergistic inhibitory effects of CDDP and 17-AAG on the growth of KYSE30 and KYSE150 cells. Cell growth and cell number were more effectively reduced by the combined treatment with CDDP and 17-AAG than by the treatment with either CDDP or 17-AAG alone. Western blotting revealed that the combined action of CDDP and 17-AAG cleaved poly (ADP-ribose) polymerase (PARP) and caspase-3, which demonstrated that the reduction in both cell growth and cell number was mediated by apoptosis. Time-course experiments showed that reduction in X-linked inhibitor of apoptosis protein (XIAP) and phosphorylated Akt were concomitant with apoptosis. The results of the present study demonstrate that 17-AAG synergizes with CDDP and induces apoptosis in CDDP-resistant ESCC cell lines, and also that modulation of the Akt/XIAP pathway may underlie this synergistic effect. Combination therapy with CDDP and an HSP90 inhibitor may represent a promising strategy to overcome CDDP resistance in ESCC.

The development and characterization of effective anticancer drugs against colorectal cancer (CRC) is of urgent need since it is the second most common cause of cancer death. The study was designed to evaluate the effects of two IGF-1R antagonists, MK-0646, a recombinant fully humanized monoclonal antibody and OSI-906, a small molecule tyrosine kinase inhibitor on CRC cells. Xenograft study was performed on IGF-1R-dependent CRC cell lines for analyzing the antitumor activity of MK-0646 and OSI-906. Tumor proliferation and apoptosis were assessed using Ki67 and TUNEL assays, respectively. We also performed in vitro characterization of MK-0646 and OSI-906 treatment on CRC cells to identify mechanisms associated with drug-induced cell death. Exposure of the GEO and CBS tumor xenografts to MK-0646 or OSI-906 led to a decrease in tumor growth. TUNEL analysis showed an increase of approximately 45-55% in apoptotic cells in both MK-0646 and OSI-906 treated tumor samples. We report the novel finding that treatment with IGF-1R antagonists led to downregulation of X-linked inhibitor of apoptosis (XIAP) protein involved in cell survival and inhibition of cell death. In conclusion, IGF-1R antagonists (MK-0646 and OSI-906) demonstrated single agent inhibition of subcutaneous CRC xenograft growth. This was coupled to pro-apoptotic effects resulting in downregulation of XIAP and inhibition of cell survival. We report a novel mechanism by which MK-0646 and OSI-906 elicits cell death in vivo and in vitro. Moreover, these results indicate that MK-0646 and OSI-906 may be potential anticancer candidates for the treatment of patients with IGF-1R-dependent CRC.

Malignant pleural mesothelioma (MPM) is a highly aggressive and conventional treatment-resistant tumor with a dismal prognosis. Among the three histological subtypes of MPM, the epithelioid is the most common type. Numb is considered as a tumor suppressor playing a critical role in controlling asymmetric cell division, maintenance of stem cell compartments, ubiquitination of specific substrates and regulating Notch-, Hedgehog- and TP53-activated pathways. The present study was designed to analyze the role of Numb in epithelioid MPM. We investigated the expression of Numb in 39 epithelioid MPM and 22 normal pleural tissues by immunohistochemistry. Furthermore, we overexpressed Numb in NCI-H2452, an epithelioid human MPM cell line, and investigated the effect of Numb overexpression on the proliferation, apoptosis and sensitivity to cisplatin in cells. The expression of Numb was significantly lower in MPM compared to the control group and Numb had an inverse correlation with the ki-67 labeling index. Loss of Numb expression was associated with poor prognosis in epithelioid MPM. Overexpression of Numb in NCI-H2452 cells significantly inhibited proliferation, promoted apoptosis and enhanced sensitivity to cisplatin. Moreover, Numb overexpression activated caspase-9 and caspase-3 through release of cytochrome c as well as downregulation of XIAP and survivin. We speculate that cytochrome c/caspase signaling is a possible mechanism through which Numb enhances the apoptosis of NCI-H2452 cells. These results suggest that Numb may be involved in epithelioid MPM development, and its upregulation may confer sensitivity to cisplatin, suggesting potential therapeutic options for MPM.

Acquired multi-drug resistance remains a major obstacle in the management of prostate cancer. The objective of this study was to examine whether chemoresistance could be due in part to the expression of the inhibitors of apoptosis proteins (IAPs). We established cisplatin-resistant LNCaP sublines. We examined the effects of cisplatin on cell growth and apoptosis in LNCaP cells and LNCaP sublines by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) assay and Hoechst 33258 staining, and analyzed cross-resistance to adriamycin, 5-fluorouracil, taxol, taxotere, and etoposide. In addition, the expression of IAP-1, IAP-2, X-linked IAP (XIAP), neuronal apoptosis inhibitory protein, and survivin was investigated by immunoblot analysis in LNCaP sublines. Although the growth rates were reduced in a dose-dependent manner by cisplatin in LNCaP sublines, the anti-proliferative effects of cisplatin were significantly decreased in LNCaP sublines compared to LNCaP cells. Cisplatin-resistant sublines, LNCaP/C1, LNCaP/ C2, and LNCaP/C3 cells, were 6.3-, 9.1-, and 22.3-fold more resistant to cisplatin than LNCaP cells, respectively, and this resistance was paralleled with reduced induction of apoptosis. LNCaP/C3 cells showed cross-resistance to adriamycin, 5-fluorouracil, and etoposide whereas those cells exhibited no or only weak cross-resistance against taxol and taxotere. With the exception of survivin, all the IAPs were identified in LNCaP cells by immunoblot analysis. Interestingly, the expression of IAP-2, XIAP, and survivin gradually increased with the extent of cisplatin-resistance. Altered expression of IAP-2, XIAP, and survivin was involved in these phenotypes of cisplatin-resistant LNCaP sublines. IAPs may make an important contribution to the resistance to the apoptotic effect of cisplatin in prostate cancer.