Anlotinib suppresses proliferation, migration, and immune escape of gastric cancer cells by activating the cGAS-STING/IFN-ß pathway.

This article reported the mechanism of Anlotinib in gastric cancer treatment. Gastric cancer cells were treated with Anlotinib (8 µM) and transfected by STING shRNA and STING vectors. Cell counting kit-8 assay, wounding healing assay, and Transwell experiment were applied for proliferation, migration, and invasion detection. PD-L1 fluorescence intensity in gastric cancer cells was explored by flow cytometry. IFN-ß level was researched by enzyme-linked immunosorbent reaction. Xenograft tumor experiment was performed by administering mice with Anlotinib and anti-PD-L1 antibody. Immunohistochemistry and western blot were used for proteins expression detection. Quantitative real-time reverse transcription-polymerase chain reaction was applied for mRNA expression detection. Hematoxylin and eosin staining was conducted on lung, liver, kidney, and cerebral cortex of mice. Gastric cancer cells treated with Anlotinib exhibited reduced proliferation, migration, and invasion (p<0.01). Anlotinib treatment reduced PCNA, CDK1, and MMP2 protein expressions and increased E-cadherin protein expression in gastric cancer cells (p<0.01). Anlotinib treatment suppressed PD-L1 expression and activated the cGAS-STING/IFN-ß pathway in gastric cancer cells (p<0.01). STING knockdown partially reversed the inhibition of Anlotinib on gastric cancer cells proliferation, migration, invasion, and immune escape (p<0.05 or p<0.01). However, STING overexpression exhibited the opposite effect. Anlotinib synergistically improved anti-tumor efficacy of anti-PD-L1 in vivo. Anlotinib synergistic anti-PD-L1 increased CD3+, CD8+ T cells, and activated the cGAS-STING/IFN-ß pathway in xenograft tumor. Anlotinib was non-toxic to lung, liver, cortex, and kidney. Anlotinib suppressed gastric cancer cells proliferation, migration, and immune escape by activating the cGAS-STING/IFN-ß pathway.

Genomic Alterations of NTRK, POLE, ERBB2, and Microsatellite Instability Status in Chinese Patients with Colorectal Cancer.

BACKGROUND: The increasing molecular characterization of colorectal cancers (CRCs) has spurred the need to look beyond RAS, BRAF, and microsatellite instability (MSI). Genomic alterations, including ERBB2 amplifications and mutations, POLE mutations, MSI, and NTRK1-3 fusions, have emerged as targets for matched therapies. We sought to study a clinically annotated Chinese cohort of CRC subjected to genomic profiling to explore relative target frequencies. METHODS: Tumor and matched whole blood were collected from 609 Chinese patients with CRC. Extracted DNA was analyzed for all classes of genomic alterations across 450 cancer-related genes, including single-nucleotide variations (SNVs), short and long insertions and deletions (indels), copy number variations, and gene rearrangements. Next-generation sequencing-based computational algorithms also determined tumor mutational burden and MSI status. RESULTS: Alterations in TP53 (76%), APC (72%), and KRAS (46%) were common in Chinese patients with CRC. For the first time, the prevalence of NTRK gene fusion was observed to be around 7% in the MSI-high CRC cohort. Across the cohort, MSI was found in 9%, ERBB2 amplification in 3%, and POLE pathogenic mutation in 1.5% of patients. Such results mostly parallel frequencies observed in Western patients. However, POLE existed at a higher frequency and was associated with large tumor T-cell infiltration. CONCLUSION: Comparing to the Western counterparts, POLE mutations were increased in our cohort. The prevalence of NTRK gene fusion was around 7% in the MSI-high CRC cohort. Increased adoption of molecular profiling in Asian patients is essential for the improvement of therapeutic outcomes. IMPLICATIONS FOR PRACTICE: The increasing use of genomic profiling assays in colorectal cancer (CRC) has allowed for the identification of a higher number of patient subsets benefiting from matched therapies. With an increase in the number of therapies, assays simultaneously evaluating all candidate biomarkers are critical. The results of this study provide an early support for the feasibility and utility of genomic profiling in Chinese patients with CRC.

Inhibition of autophagy in hepatocarcinoma cells promotes chemotherapeutic agent-induced apoptosis during nutrient deprivation.

Autophagy is a lysosome-dependent process involved in protein and organelle degradation. It has been suggested that autophagy is activated in nutrient-deficient condition and plays an important role in protecting cells from nutrient shortage. However, the effect of autophagy on chemotherapy during nutrient deficiency has been rarely researched. In the present study, we discovered that hepatocarcinoma cells exhibit chemoinsensitivity accompanied by the activation of autophagy when cultured in nutrient-deprived medium. Inhibition of autophagy by 3-methyladenine or siRNA­targeted Beclin 1 increased the nutrient deprivation­induced apoptosis and chemosensitivity in hepatocarcinoma cells. Furthermore, decreased mitochondrial mass was detected when cells underwent autophagy. The present study suggests that induction of autophagy confers a survival advantage for hepatocarcinoma cells during nutrient deprivation, not only rescuing cells from nutrient deficiency-induced cell apoptosis, but also protecting cells from chemotherapy-induced cell death. Combined usage of the inhibition of autophagy and conventional chemotherapeutic agents could be an effective therapy for hepatocarcinoma during nutrient deprivation.

Docetaxel-mediated autophagy promotes chemoresistance in castration-resistant prostate cancer cells by inhibiting STAT3.

Signal transducer and activator of transcription (STAT)3 expression is correlated with neoplasm growth, metastasis, and prognosis; it has also been implicated in the regulation of autophagy, which may in turn contribute to tumor chemoresistance. However, it is unknown whether STAT3 is involved in cancer cell survival in response to chemotherapy. In this study, we show that autophagy is triggered during chemotherapy and that inhibiting autophagy increased chemosensitivity of castration-resistant prostate cancer (CRPC) cells. Meanwhile, docetaxel induced autophagy was inhibited by STAT3 activation, which increased mitochondrial damage and decreased CRPC cell viability. These results suggest that STAT3 contributes to CRPC cell survival and chemoresistance by modulating autophagy.

Suppression of p53 potentiates chemosensitivity in nutrient-deprived cholangiocarcinoma cells via inhibition of autophagy.

Tumor protein p53 has been intensively studied as a major tumor suppressor. The activation of p53 is associated with various anti-neoplastic functions, including cell senescence, cell cycle arrest, apoptosis and inhibition of angiogenesis. However, the role of p53 in cancer cell chemosensitivity remains unknown. Cholangiocarcinoma cell lines QBC939 and RBE were grown in full-nutrient and nutrient-deprived conditions. The cell lines were treated with 5-fluorouracil or cisplatin and the rate of cell death was determined in these and controls using Cell Counting Kit-8 and microscopy-based methods, including in the presence of autophagy inhibitor 3MA, p53 inhibitor PFT-α or siRNA against p53 or Beclin-1. The present study demonstrated that the inhibition of p53 enhanced the sensitivity to chemotherapeutic agents in nutrient-deprived cholangiocarcinoma cells. Nutrient deprivation-induced autophagy was revealed to be inhibited following inhibition of p53. These data indicate that p53 is important for the activation of autophagy in nutrient-deprived cholangiocarcinoma cells, and thus contributes to cell survival and chemoresistance.

Inhibition of p53 increases chemosensitivity to 5-FU in nutrient-deprived hepatocarcinoma cells by suppressing autophagy.

Activation of p53 can induce apoptosis, cell cycle arrest, and cell senescence, although some evidence has suggested that p53 could promote cell survival. However, whether p53 plays a positive role in cancer cell survival to chemotherapy remains unknown. In this study, we show that inhibition of p53 enhanced apoptosis and increased chemosensitivity to 5-fluorouracil (5-FU) in nutrient-deprived hepatocarcinoma cells (HCC). Meanwhile, nutrient-deprivation-induced autophagy was inhibited by pifithrin-α or small interfering RNA targeting p53. The expression of p53 was not increased when HCC were incubated under nutrient-deprived conditions. This indicates that the basal level of p53 is important to autophagy activation in nutrient-deprived HCC cells. Furthermore, combining p53 inhibition and nutrient deprivation or 5-FU treatment resulted in a marked increase in reactive oxygen species generation and mitochondrial damage. Antioxidants reduced nutrient deprivation or 5-FU-induced cell death of HCC after p53 inhibition. Our results suggest that p53 contributes to cell survival and chemoresistance in HCC under nutrient-deprived conditions by modulating autophagy activation.

Autophagy contributes to the survival of CD133+ liver cancer stem cells in the hypoxic and nutrient-deprived tumor microenvironment.

Liver cancer stem cells (LCSCs) can drive and maintain hepatocellular carcinoma (HCC) growth, metastasis, and recurrence. Therefore, they are potentially responsible for the poor prognosis of HCC. Oxygen and nutrient deficiencies are common characteristics of the tumor microenvironment. However, how LCSCs adapt to oxygen- and nutrient-deprived conditions is unclear. Here, we used immunofluorescent staining and flow cytometry analysis to show that CD133+ cells were significantly enriched after hypoxia and nutrient starvation (H/S) in the human HCC cell line Huh7. Sorted CD133+ cells showed higher survival, less apoptosis, and possess higher clonogenic ability under H/S compared to the CD133- population. Under H/S, electron microscopy revealed more advanced autophagic vesicles in CD133+ cells. Additionally, CD133+ cells had higher autophagy levels as measured by both RT-qPCR and Western blotting. CD133+ cells had more accumulated GFP-LC3 puncta, which can be detected by fluorescence microscopy. The autophagic inhibitor chloroquine (CQ) significantly increased apoptosis and decreased the clonogenic capacity of CD133+ cells under H/S. Pre-culturing in H/S enhanced the sphere-forming capacity of CD133+ cells. However, CQ significantly impaired this process. Therefore, autophagy is essential for LCSCs maintenance. CD133+ cells were also found to have a higher tumor-forming ability in vivo, which could be inhibited by CQ administration. Collectively, our results indicate that the involvement of autophagy in maintenance of CD133+ LCSCs under the oxygen- and nutrient-deprived conditions that are typical of the tumor microenvironment in HCC. Therefore, autophagy inhibitors may make LCSCs more sensitive to the tumor microenvironment and be useful in improving anti-cancer treatments.

Inhibition of autophagy enhances anticancer effects of bevacizumab in hepatocarcinoma.

Angiogenesis inhibitors have long been considered desirable anticancer agents. However, it was found that many tumors could develop resistance to antiangiogenesis inhibitors. Antiangiogenic therapy results in metabolic stress. Autophagy is an important survival mechanism in cancer cells under metabolic stress; however, it remains unknown if autophagy contributes to antiangiogenesis resistance. In this study, we reported that bevacizumab treatment reduced the development of new blood vessels and inhibited cell growth in xenografts of hepatocellular carcinoma (HCC) tumors. Bevacizumab treatment also upregulated expression of the autophagy-related genes (Beclin1 and LC3) and increased autophagosome formation. Our in vitro studies demonstrated that autophagy inhibition significantly increased apoptosis of HCC cells during nutrient starvation or hypoxia. In addition, the combined treatment of an autophagy inhibitor and bevacizumab markedly inhibited the tumor growth of HCC xenografts, led to enhanced apoptosis, and impaired the proliferation of tumor cells compared with treatment with either drug alone. Furthermore, autophagy inhibition led to enhanced reactive oxygen species (ROS) generation in HCC cells exposed to nutrient starvation or hypoxia in vitro and increased DNA oxidative damage in vivo. Antioxidants reduced nutrient starvation or the hypoxia-induced cell death of HCC cells after autophagy inhibition. Our results suggest that autophagy modulates ROS generation and contributes to cell survival under metabolic stress. Therefore, autophagy inhibition may be a novel way of increasing the efficicacy of antiangiogenic agents in the treatment of HCC.

Anti-tumor effect of 5-aza-2'-deoxycytidine by inhibiting telomerase activity in hepatocellular carcinoma cells.

AIM: To investigate the effect of the demethylating reagent 5-aza-2'-deoxycitidine (DAC) on telomerase activity in hepatocellular carcinoma (HCC) cell lines, SMMC-7721 and HepG2. METHODS: The related gene expression in cell lines was examined by real-time reverse transcription-polymerase chain reaction and Western blotting analysis. The telomerase activity was examined by telomeric repeat amplification protocol-enzyme-linked immunosorbent assay and DNA methylation was determined by methylation-specific polymerase chain reaction. RESULTS: The telomerase activity was significantly reduced in both cell lines treated with DAC, accompanied by downregulation of telomerase reverse transcriptase (hTERT). We also observed the effect of DAC on the methylation status of hTERT promoter and the expression of regulatory genes, such as c-myc, p15, p16, p21, E2F1, and WT1. The methylation status of hTERT promoter could be reversed in SMMC-7721 by DAC, but not in HepG2 cells. However, p16 expression could be reactivated by demethylation of its promoter, and c-Myc expression was repressed in both cell lines. Moreover, DAC could enhance the sensitivity to the chemotherapeutic agents, such as cisplatin, by induction of apoptosis of HCC cells. CONCLUSION: The DAC exerts its anti-tumor effects in HCC cells by inhibiting the telomerase activity.

Targeting autophagy potentiates chemotherapy-induced apoptosis and proliferation inhibition in hepatocarcinoma cells.

Induction of cell death and inhibition of cell growth are the main targets of cancer therapy. Here we evaluated the role of autophagy on chemoresistance of human hepatocarcinoma (HCC) cell lines, focusing on its crosstalk with cell apoptosis and proliferation. In this study, a chemotherapeutic agent (cisplatin or 5FU) induced the formation of autophagosomes in three human HCC cell lines and upregulated the expression of autophagy protein LC3-II. Inhibition of autophagy by 3-methyladenine or si-beclin 1 increased chemotherapy-induced apoptosis in HCC cells. Meanwhile, increased damage of the mitochondrial membrane potential was also observed in HCC cells when autophagy was inhibited. Furthermore, inhibition of autophagy reduced clone formation and impaired cell growth of HCC cells when treated with chemotherapy. Co-administration of an autophagy inhibitor (chloroquine) and chemotherapy significantly inhibited tumor growth in a mouse xenograft tumor model, with greater extent of apoptosis and impaired proliferation of tumor cells. This study suggests that autophagy is a potential novel target to improve therapy efficiency of conventional chemotherapeutics towards HCC.

Autophagic cell death induced by 5-FU in Bax or PUMA deficient human colon cancer cell.

Autophagy is a membrane process that results in the transporting of cellular contents to lysosomes for degradation. Autophagic cell death is another way of programed cell death called type II PCD, which has complicated connection with apoptosis, both of these two types of cell death play an important role in tumor development. In this study, we investigated chemotherapeutic agent induced cell death pathway in wild type (WT), Bax(-/-) and PUMA(-/-) HCT116 cells. Bax or PUMA deficient cells had similar chemosensitivity to WT cells but were defective in undergoing apoptosis. The results of electron microscopy and GFP-LC3 localization assay showed that autophagy was induced in Bax or PUMA deficient cells but not in WT cells. mTOR activity was decreased in Bax or PUMA deficient cells which further indicated the up-regulation of autophagy. Inhibition of autophagy by 3-Methyladenine (3-MA) decreased the cell death in Bax or PUMA deficient cells. Taken together, these results suggest that autophagic cell death can be used as an alternative cell death pathway in apoptosis defective cells and may bring a new target for cancer therapy.

Up-regulation of hTERT expression by low-dose cisplatin contributes to chemotherapy resistance in human hepatocellular cancer cells.

The telomerase is specifically activated in most malignant tumors but is usually inactive in normal somatic cells. It has been reported that telomerase has an anti-apoptotic role and up-regulation of telomerase helps cancer cells to be resistant to chemotherapeutic agent-induced cell death. The effect of cisplatin on telomerase activity is complex, and the exact mechanism remains largely unknown. In this study, we found that cisplatin activated telomerase activity and human telomerase reverse transcriptase (hTERT) expression in SMMC7721 human hepatocellular carcinoma cell line. Low-dose cisplatin up-regulated hTERT and NF-kappaB p65 expression and increased telomerase and NF-kappaB activity. Inhibition of NF-kappaB attenuated the hTERT expression and telomerase activity exposed to cisplatin, suggesting that NF-kappaB is responsible for the cisplatin-induced activation of the hTERT. Furthermore, preincubation of low-dose cisplatin which induced high expression of hTERT help hepatocellular carcinoma SMMC7721 cells survive under the high concentration of anti-cancer drugs. Inhibition of hTERT increased sensitivity of SMMC7721 cells to chemotherapy. Taken together, these results suggested that up-regulation of hTERT expression by low-dose cisplatin is NF-kappaB-dependent and contributes to chemotherapy resistance in human hepatocellular cancer cells.

Protective effect of estrogen on apoptosis in a cell culture model of Parkinson's disease.

OBJECTIVES: The protective effect of estrogen on the neurons in Parkinson's disease (PD) is unclear. The present study aimed to investigate the effect of estrogen on the apoptosis and dopaminergic function on a cultured cell model of PD. METHODS: The PD model was established by addition of 1-methyl-4-phenylpyridinium (MPP+) to PC12 cell culture. Estrogen was added to cell groups with MPP+ (Estrogen+MPP+), and without MPP+ (Estrogen only group). Cell viability, content of tyrosine hydroxylase (TH), apoptosis ratio, expression of apoptosis-suppression protein Bcl-x and apoptosis-acceleration protein IL-1 beta converting enzyme (ICE) were measured. RESULTS: Cell viability in the Estrogen+MPP+ group was similar to the control group but was higher than in the MPP+ group (P < 0.05). The apoptosis ratios in the Estrogen+MPP+ group (33.6%), and the control group (31.3%), were also similar, but it was lower than in the MPP+ group (63.5%, P < 0.05). Concentrations of Bcl-x were higher in the Estrogen+MPP+ group, whereas ICE concentrations were lower than in the MPP+ group (P < 0.05). CONCLUSIONS: Estrogen suppresses apoptosis and improves cell viability in MPP+ induced injuries in the PC12 cells. The beneficial effects of estrogen on the PD model are due to the suppression of pro-apoptotic protein ICE, and stimulation of anti-apoptotic protein Bcl-x.

Lentiviral vector-mediated siRNA knockdown of SR-PSOX inhibits foam cell formation in vitro.

AIM: To investigate the expression of scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXC chemokine ligand 16 (CXCL16) in the human monocyte-derived cell line THP-1, and the effect of lentiviral vectors for the stable delivery of SR-PSOX/CXCL16 short hairpin RNA on foam cell formation. METHODS: A lentiviral expression vector containing enhanced green fluorescence protein (GFP) and SR-PSOX small interfering RNA (siRNA) (Lenti-SR-PSOXsi), or the control siRNA (Lenti-NC) gene was constructed. A human monocyte-derived cell line THP-1 was transfected with a different multiplicity of infection (MOI) of Lenti-SR-PSOXsi or Lenti-NC, and cultured to obtain stably-transfected THP- 1KD and THP-1NC cells. After incubation with oxidatively-modified, low-density lipoprotein (Ox-LDL), the expression of SR-PSOX/CXCL16 mRNA was determined by real-time PCR. The expression of the SR-PSOX/CXCL16 protein was detected by flow cytometry analysis. The effect of Lenti-SR-PSOXsi on foam cell formation was assessed by Oil red O-stain analysis. RESULTS: Ox-LDL increased the expression of SR-PSOX/CXCL16 mRNA in a time- and dose-dependent manner in THP-1 cells. Four days after transfection with Lenti-SR-PSOXsi (MOI: 100), the percentage of GFP expression cells was over 89.3%. The expression of the SR-PSOX/ CXCL16 mRNA and protein in THP-1KD cells significantly decreased compared with the parent cells, even the THP-1KD cells stimulated with 40 mg/L Ox-LDL. Ox-LDL uptake experiments in THP-1- and THP-1KD-derived macrophages indicated that SR-PSOX/CXCL16 deficiency decreased the development of macrophage- derived foam cell formation. CONCLUSION: The above data showed that SRPSOX siRNA delivered by using lentiviral vectors in THP-1 cells was a powerful tool for studying the effect of SR-PSOX, and decreased the expression of the SRPSOX gene by inhibiting macrophage-derived foam cell formation.