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Background: Many studies have reported that the inflammatory immune response related to TLR9 signaling activation participates in tumor development and affects the treatment outcome. RUNX3 functions as a tumor suppressor by regulating DNA methylation. RUNX3 protein plays an important role in TGF-β signaling pathway that is involved in tumor growth inhibition and apoptosis. At present, radiotherapy is still an important treatment in lung cancer, which induces immune response and affects the therapeutic outcome. The role of TLR9 signaling activation and RUNX3 in this process is not clear., Methods: In this study, we investigated the expression of TLR9 in tumor and RUNX3 in surrounding tissues by immunohistochemical methods and analyzed the relationship on postoperative survival in lung cancer., Results: We found that the high expression of TLR9 was the risk factor in postoperative survival of lung cancer with no difference in lifetime. The high expression of RUNX3 in lung cancer with TLR9 signaling activation was in favor of progression-free survival and overall survival in postoperative radiotherapy. It suggested that RUNX3 played an important role in lung cancer radiotherapy. In order to determine the effect of RUNX3 in lung cancer radiation with TLR9 signaling activation, we introduced 5-Aza-2'-deoxycytidine (5-Aza-CdR) and exposed lung cancer A459 cells repeatedly. The high expression of RUNX3 especially RUNX3 -B in cells treated with 5-Aza-CdR was observed. We examined that 5-Aza-CdR induced more cell blocking in G2/M phase in combining irradiation., Conclusion: The result implied that it was feasible to improve radiosensitivity of lung cancer with TLR9 signaling activation by increasing RUNX3 expression, and 5-Aza-CdR was an option in this process., Competing Interests: The authors report no competing interests in this work., (© 2021 Gao et al.)

Background: The relationship of inflammation and tumor is becoming more and more important in the study on the pathogenesis of colorectal cancer. The role of TLR9-mediated immune inflammation reaction in the process is not currently clear. The purpose of the study was to discuss the correlation of TLR9 signal activation with tumor progression by detecting the expression of TLR9 and its downstream molecule NF-kappaB in colorectal cancer tissues at different stages., Methods: TLR9 expression in colorectal cancer tissues was detected by immunohistochemical streptavidin-perosidase method and Western blot., Results: The result showed that the high expression of TLR9 was correlated with tumor poorly differentiation, invasion and liver metastasis, the abnomal increasing levels of CEA in blood. With the signal activation, the levels of TLR9 protein raised more in advanced colorectal cancer than in early colorectal cancer. Afterward, we found that the activation of specific expression of TLR9 signal was related to histologic origin. TLR9-C expression displayed in both advanced cancer and para-carcinoma tissues, and TLR9-R protein was predominat in partial sigmoid and rectal cancer tissues. With the differential expression of TLR9, the levels of its downstream molecule NF-kappaB protein increased in colon cancer tissues and decreased in rectal cancer tissues., Conclusion: The results confirmed that TLR9 signaling activation participated in the clinical process of colorectal cancer and influenced NF-kappaB expression., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background: The beneficial antitumor effects of low-molecular-weight heparins (LMWHs) have previously been investigated in basic and clinical studies. In this study, the antitumor efficacy of nadroparin combined with radiotherapy was investigated in vivo., Methods: A total of 48 tumor-bearing mice were randomly divided into six groups (n=8 per group): control group, irradiation group (X), LMWH 1,000 group, LMWH 2,000 group, LMWH 1,000 +X group and LMWH 2,000 +X group. Following this, tumor growth, weight and inhibitory rate, as well as the survival of mice in each group, were determined. Levels of serum interleukin (IL)-6 and transforming growth factor (TGF)-β1 were determined via enzyme-linked immunosorbent assay (ELISA) analyses. The expression levels of CD34 were investigated using immunohistochemistry analyses to represent the microvascular density (MVD) values of tumor tissues. In addition, tumor cell apoptosis was investigated using TdT-mediated dUTP nick end labeling (TUNEL) analysis post treatment. The expression levels of survivin were analyzed by Western blotting., Results: The volumes and weights of tumors in the treatment groups were demonstrated to be significantly decreased, which was most obvious in the LMWH 2,000 +X group. The tumor inhibitory rate was significantly increased in the treated mice. ELISA assays demonstrated that the concentrations of serum IL-6 and TGF-β1 were significantly decreased in the LMWH 2,000 +X group. In addition, the decreased CD34 expression was found in the combined treatment groups. TUNEL assays demonstrated that the apoptosis rate was increased in treated mice, and the highest apoptosis rate was exhibited by the LMWH 2,000 +X group. Results of Western blotting demonstrated that combinatory treatment with both nadroparin and X-ray irradiation significantly inhibited the expression of survivin., Conclusion: These results demonstrated that a combinatory treatment strategy of nadroparin with fractionated irradiation had a strong synergistic antitumor effect in vivo, which may be associated with the promotion of apoptosis, inhibited secretion of TGF-β1 and IL-6 and down-regulation of CD34 and survivin expression., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background: The combined therapy of bevacizumab (BEV) with taxane (paclitaxel or docetaxel) has shown an improvement on progression-free survival (PFS) and objective remission in Her2-negative patients with locally recurrent or metastatic breast cancer (LR/MBC). However, there was no benefit in overall survival (OS). The aim of this study was to evaluate the efficacy and safety of adding an agent to the BEV/taxane regimens for the treatment of Her2-negative patients with LR/MBC in a first-line setting., Materials and Methods: We searched PubMed, Web of Science, EMBASE, EBSCO, and the Cochrane Library databases for eligible trials. A meta-analysis was performed using Review Manager 5.0 freeware package. We calculated the hazard ratio (HR) for PFS and OS. The odds ratio (OR) was used to calculate objective response rate (ORR) and grade 3/4 drug-related adverse events. The heterogeneity of study outcomes was calculated by the χ (2) test or I (2) statistics., Results: A total of 1,124 patients from seven randomized controlled trials were analyzed. Our meta-analysis showed that the ORR was significantly improved in the BEV/taxane-based triplet group when compared with the BEV/taxane-based doublet group (OR =1.31, 95% confidence interval [CI]: 1.03-1.67, P=0.03). A subset analysis showed that a similar result was achieved in the triplet group in which a cytotoxic agent was added (OR =1.46, 95% CI: 1.09-1.95, P=0.01). However, the PFS and OS had no statistically significant differences between the two groups (HR =0.87, 95% CI: 0.68-1.13, P=0.31; HR =0.98, 95% CI: 0.82-1.16, P=0.78, respectively). Regarding safety, thromboembolic events, fatigue, and diarrhea (all $grade 3) were more frequently observed in the BEV/taxane-based triplet group (OR =3.8, 95% CI: 1.86-7.79, P=0.0003; OR =1.55, 95% CI: 1.05-2.27, P=0.03; OR =2.1, 95% CI: 1.29-3.41, P=0.003, respectively). Other toxic effects had no statistically significant differences between the two groups., Conclusion: Our results showed that adding an agent to BEV/taxane treatment regimens did not significantly improve PFS and prolong OS, except for conferring a significant advantage toward improved ORR in the first-line therapy for Her2-negative patients with LR/MBC. However, its side effects are predictable and manageable.

Radiotherapy is a powerful cure for local advanced non-small cell lung cancer. However, radioresistance and tumor relapse still occur in a high proportion of patients. Octamer-4 (Oct-4), a transcription factor of the POU family, plays a key role in maintaining chemoradioresistant properties and regulating cancer progression. In this study, we demonstrated that Oct-4 expression was significantly increased in radioresistant H460 (H460R) cell line. CpG oligodeoxyribonucleotide (CpG-ODN) 7909 sensitized H460R cells when combined with irradiation treatment. The clonogenic capacity was significantly decreased, and the values of D0 and Dq were lower than those of irradiation alone group. The sensitive enhancement ratio (SER) of D0 was 1.224. This combined treatment led to a dramatic reduction in Oct-4 expression in a dose-dependent manner and also showed increased percentage of cells in the radiosensitive G2/M phase relative to either treatment alone. These results identified that Oct-4 was involved in radioresistance. CpG-ODN 7909 could enhance radiosensitivity partly through downregulating Oct-4 expression in radioresistant lung cancer cells.

Objective: The aim of the study reported here was to further investigate the potential effect of ataxia-telangiectasia mutated (ATM) kinase-dependent hyperphosphorylation of checkpoint kinase-2 (Chk2) on radiosensitivity enhanced by oligodeoxynucleotide 7909 containing CpG motifs (CpG ODN7909) in human lung adenocarcinoma A549 cells., Methods: In vitro A549 cells were randomly separated into control, CpG, X-ray, CpG+ X-ray, ATM kinase-small interfering RNA (siRNA)+CpG+X-ray (ATM-siRNA), and Chk2-siRNA+CpG+X-ray (Chk2-siRNA) groups. siRNAs were adopted to silence the ATM and Chk2 genes. Expression and phosphorylation of ATM kinase and Chk2 were detected by Western blot assay. Cell colonies were observed under inverted phase-contrast microscopy. Cellular survival curves were fitted using a multi-target single-hitting model. Cell cycle and apoptosis were analyzed by flow cytometry., Results: Expression of ATM kinase and Chk2 was similar among the control, CpG, X-ray, and CpG+X-ray groups. Phosphorylated ATM kinase and Chk2 were significantly increased in the CpG+X-ray group compared with in the X-ray group (t=6.00, P<0.01 and t=3.13, P<0.05, respectively), though these were hardly detected in the control and CpG groups. However, expression of ATM kinase and Chk2 was clearly downregulated in the ATM-siRNA and Chk2-siRNA groups, respectively. Similarly, their phosphorylation levels were also significantly decreased in the ATM-siRNA group (t=14.35, P<0.01 and t=8.46, P<0.01, respectively) and a significant decrease in phosphorylated Chk2 was observed in the Chk2-siRNA group (t=7.28, P<0.01) when compared with the CpG+X-ray group. Further, the number of A549 cells at Gap 2/mitotic phase and the apoptosis rate were clearly increased in the CpG+X-ray group compared with in the other groups (all P<0.05). The multi-target single-hitting model showed that the sensitization enhancement ratio calculated by mean death dose was 1.39 in CpG+X-ray group (vs 1.04 and 1.03 in the ATM-siRNA and Chk2-siRNA groups, respectively)., Conclusion: This study provides the first evidence, as far as we are aware, that CpG ODN7909 can potentiate A549 cell radiosensitivity via increasing ATM kinase-dependent phosphorylation of Chk2, suggesting activation of the ATM kinase/Chk2 signal pathway. However, the mechanism of ATM kinase activation is worth further exploration.

Background: Cytosine-phosphate-guanine (CpG) oligodeoxyribonucleotides (ODNs), which induce signaling via Toll-like receptor 9, have recently been suggested to enhance sensitivity to traditional therapies, including chemotherapy, in certain cancer cell lines. This study aimed to define the dose-effect relationship for CpG ODN 1826 in increasing radiosensitivity and its impact on immune function in a mouse model of Lewis lung cancer., Methods: The tumor-bearing mouse model was induced by injecting Lewis lung cancer cells into the right anterior leg subcutaneously. Sixty-four C57BL/6 J mice were evenly randomized into eight groups, comprising: a control group; an irradiation group; a CpG ODN 0.15 group; a CpG ODN 0.3 group; a CpG ODN 0.45 group; a CpG 0.15 + irradiation group; a CpG 0.3 + irradiation group; and a CpG 0.45 + irradiation group. Tumor growth, serum tumor necrosis factor-alpha and interleukin-12 concentrations, spleen and thymus exponents, and effect of CpG on the secondary immune response were measured, and apoptosis of tumor cells was investigated using TdT-mediated dUTP nick end labeling (TUNEL) after treatment., Results: Tumor volumes in the treated groups were smaller than in the control group, with those of the CpG 0.45 + irradiation group being the smallest. TUNEL showed that the apoptosis rate in all the active treatment groups was higher than in the control group. CpG ODN apoptosis rate, serum tumor necrosis factor-alpha and interleukin-12 levels, and the spleen and thymus exponent showed greater improvement in the groups receiving combination therapy of CpG ODN and irradiation than the control group or the group receiving irradiation alone. With the increasing concentration of CpG ODN 1826, its effect became more and more significant, meanwhile, inoculation of Lewis lung cancer cells failed in those CpG ODN-cured mice., Conclusion: CpG ODNs dramatically increased the radiosensitivity of Lewis lung cancer and enhanced immune function in mice in a dose-related manner.

Objective: To investigate the impact of checkpoint kinase 2 (CHK2)-small interfering RNA (CHK2-siRNA) on the enhancement of radiosensitivity by CpG oligodeoxynucleotide (ODN) 7909 in lung cancer A549 cells., Methods: The A549 cells were randomly divided into five groups: control, CpG, X-ray, CpG + X-ray, and CHK2-siRNA + CpG + X-ray. Cell colonization was observed using inverted microscopy. Cell cycle and apoptosis were analyzed by flow cytometry. CHK2 expression was detected by Western blot. CHK2-siRNA was adopted to silence the expression of CHK2., Results: The level of CHK2 phosphorylation was higher in the CpG + X-ray group than in the X-ray group. Increases in G(2)/mitotic (M) phase arrest and apoptosis and a decrease of cell survival rate in the CpG + X-ray group were statistically significant (P < 0.05) when compared with the CHK2-siRNA + CpG + X-ray group in which the expression of CHK2 was obviously inhibited. The combination of CpG ODN7909 and X-ray irradiation was found to enhance the mitotic death of A549 cells. The sensitization enhancement ratio of mean death dose (D(0)) was 1.42 in the CpG + X-ray group, which was higher than that of the CHK2-siRNA + CpG + X-ray group, in which D(0) was 1.05., Conclusion: To a certain extent, the impact of a combination of CpG ODN7909 and X-ray on G(2)/M phase arrest, apoptosis, and rate of cell survival was attenuated by CHK2-siRNA in human lung adenocarcinoma A549 cells, indicating that increased phosphorylation of CHK2 might be a radiosensitive pathway.