Inhibition of autophagy potentiates the antitumor effect of the multikinase inhibitor sorafenib in hepatocellular carcinoma

Multikinase inhibitor sorafenib inhibits proliferation and angiogenesis of tumors by suppressing the Raf/MEK/ERK signaling pathway and VEGF receptor tyrosine kinase. It significantly prolongs median survival of patients with advanced hepatocellular carcinoma (HCC) but the response is disease-stabilizing and cytostatic rather than one of tumor regression. To examine the mechanisms underlying the relative resistance in HCC, we investigated the role of autophagy, an evolutionarily conserved self-digestion pathway, in hepatoma cells in vitro and in vivo. Sorafenib treatment led to accumulation of autophagosomes as evidenced by conversion from LC3-I to LC3-II observed by immunoblot in Huh7, HLF and PLC/PRF/5 cells. This induction was due to activation of autophagic flux, as there was further increase in LC3-II expression upon treatment with lysosomal inhibitors, clear decline of the autophagy substrate p62, and an mRFP-GFP-LC3 fluorescence change in sorafenib-treated hepatoma cells. Sorafenib inhibited the mammalian target of rapamycin complex 1 and its inhibition led to accumulation of LC3-II. Pharmacological inhibition of autophagic flux by chloroquine increased apoptosis and decreased cell viability in hepatoma cells. siRNA-mediated knockdown of the ATG7 gene also sensitized hepatoma cells to sorafenib. Finally, sorafenib induced autophagy in Huh7 xenograft tumors in nude mice and coadministration with chloroquine significantly suppressed tumor growth compared with sorafenib alone. In conclusion, sorafenib administration induced autophagosome formation and enhanced autophagic activity, which conferred a survival advantage to hepatoma cells. Concomitant inhibition of autophagy may be an attractive strategy for unlocking the antitumor potential of sorafenib in HCC.     

NAC1 modulates sensitivity of ovarian cancer cells to cisplatin by altering the HMGB1-mediated autophagic response

Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, is known to have important roles in proliferation and growth of tumor cells and in chemotherapy resistance. Yet, the mechanisms underlying how NAC1 contributes to drug resistance remain largely unclear. We report here that autophagy was involved in NAC1-mediated resistance to cisplatin, a commonly used chemotherapeutic drug in the treatment of ovarian cancer. We found that treatment with cisplatin caused an activation of autophagy in ovarian cancer cell lines, A2780, OVCAR3 and SKOV3. We further demonstrated that knockdown of NAC1 by RNA interference or inactivation of NAC1 by inducing the expression of a NAC1 deletion mutant that contains only the BTB/POZ domain significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity. Moreover, inhibition of autophagy and sensitization to cisplatin by NAC1 knockdown or inactivation were accompanied by induction of apoptosis. To confirm that the sensitizing effect of NAC1 inhibition on the cytotoxicity of cisplatin was attributed to suppression of autophagy, we assessed the effects of the autophagy inhibitors 3-methyladenosine and chloroquine, and small interfering RNAs (siRNAs) targeting beclin 1 or Atg5 on the cytotoxicity of cisplatin. Treatment with 3-methyladenosine, chloroquine or beclin 1 and Atg5-targeted siRNA also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin. Regulation of autophagy by NAC1 was mediated by the high-mobility group box 1 (HMGB1), as the functional status of NAC1 was associated with the expression, translocation and release of HMGB1. The results of our study not only revealed a new mechanism determining cisplatin sensitivity but also identified NAC1 as a novel regulator of autophagy. Thus, the NAC1-mediated autophagy may be exploited as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.     

p53-regulated autophagy is controlled by glycolysis and determines cell fate

The tumor suppressor p53 regulates downstream targets that determine cell fate. Canonical p53 functions include inducing apoptosis, growth arrest, and senescence. Non-canonical p53 functions include its ability to promote or inhibit autophagy and its ability to regulate metabolism. The extent to which autophagy and/or metabolic regulation determines cell fate by p53 is unclear. To address this, we compared cells resistant or sensitive to apoptosis by the p53 activator Nutlin-3a. In resistant cells, glycolysis was maintained upon Nutlin-3a treatment, and activated p53 promoted prosurvival autophagy. In contrast, in apoptosis sensitive cells activated p53 increased superoxide levels and inhibited glycolysis through repression of glycolytic pathway genes. Glycolysis inhibition and increased superoxide inhibited autophagy by repressing ATG genes essential for autophagic vesicle maturation. Inhibiting glycolysis increased superoxide and blocked autophagy in apoptosis-resistant cells, causing p62-dependent caspase-8 activation. Finally, treatment with 2-DG or the autophagy inhibitors chloroquine or bafilomycin A1 sensitized resistant cells to Nutlin-3a-induced apoptosis. Together, these findings reveal novel links between glycolysis and autophagy that determine apoptosis-sensitivity in response to p53. Specifically, the findings indicate 1) that glycolysis plays an essential role in autophagy by limiting superoxide levels and maintaining expression of ATG genes required for autophagic vesicle maturation, 2) that p53 can promote or inhibit autophagy depending on the status of glycolysis, and 3) that inhibiting protective autophagy can expand the breadth of cells susceptible to Nutlin-3a induced apoptosis.     

In vivo antitumor activity of interleukin 21 mediated by natural killer cells

Immunotherapy with high-dose interleukin (IL) 2 has been shown to successfully treat tumors in animal models and cause dramatic tumor regressions in some patients with metastatic melanoma, renal cell carcinoma, and non-Hodgkin's lymphoma. However, toxicity associated with IL-2 administration has compromised its widespread use in the clinic. IL-21 is a more recently discovered cytokine produced by activated CD4(+) T cells that shares significant sequence homology to IL-2, IL-4, and IL-15. Because IL-21 and IL-2 and their receptors share significant sequence similarities and both cytokines can stimulate T and natural killer (NK) cells, we sought to study whether IL-21, like IL-2, exhibits antitumor effects in vivo. In this study, we treated established s.c. tumor in mice by systemically administering plasmid DNA encoding murine IL-21 using a hydrodynamics-based gene delivery technique. Administration of IL-21 plasmid DNA resulted in high levels of circulating IL-21 in vivo. Treatment of tumor-bearing mice with IL-21 plasmid DNA significantly inhibited the growth of B16 melanoma and MCA205 fibrosarcoma in a dose-dependent manner without significant toxicity and increased the survival rate, compared with mice treated with control plasmid DNA. In vivo depletion of either CD4(+) or CD8(+) T cells did not affect IL-21-mediated antitumor activity. However, depletion of NK cells completely abolished IL-21-induced tumor inhibition. Consistent with this, the antitumor activity of IL-21 seemed to be mediated through enhanced cytolytic activity of NK cells. Our study suggests that IL-21 has significant antitumor activity and may have therapeutic potentials as an antitumor agent in the clinic.     

Synergistic combination therapy with nanoliposomal C6-ceramide and vinblastine is associated with autophagy dysfunction in hepatocarcinoma and colorectal cancer models

Autophagy, a catabolic survival pathway, is gaining attention as a potential target in cancer. In human liver and colon cancer cells, treatment with an autophagy inducer, nanoliposomal C6-ceramide, in combination with the autophagy maturation inhibitor, vinblastine, synergistically enhanced apoptotic cell death. Combination treatment resulted in a marked increase in autophagic vacuole accumulation and decreased autophagy maturation, without diminution of the autophagy flux protein P62. In a colon cancer xenograft model, a single intravenous injection of the drug combination significantly decreased tumor growth in comparison to the individual treatments. Most importantly, the combination treatment did not result in increased toxicity as assessed by body weight loss. The mechanism of combination treatment-induced cell death both in vitro and in vivo appeared to be apoptosis. Supportive of autophagy flux blockade as the underlying synergy mechanism, treatment with other autophagy maturation inhibitors, but not autophagy initiation inhibitors, were similarly synergistic with C6-ceramide. Additionally, knockout of the autophagy protein Beclin-1 suppressed combination treatment-induced apoptosis in vitro. In conclusion, in vitro and in vivo data support a synergistic antitumor activity of the nanoliposomal C6-ceramide and vinblastine combination, potentially mediated by an autophagy mechanism.     

A Switch Between Cytoprotective and Cytotoxic Autophagy in the Radiosensitization of Breast Tumor Cells by Chloroquine and Vitamin D

Calcitriol or 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, as well as vitamin D analogs, has been shown to increase sensitivity to ionizing radiation in breast tumor cells. The current studies indicate that the combination of 1,25-dihydroxyvitamin D3 with radiation appears to kill p53 wild-type, estrogen receptor-positive ZR-75-1 breast tumor cells through autophagy. Minimal apoptosis was observed based on cell morphology by DAPI and TUNEL staining, annexin/PI analysis, caspase-3, and PARP cleavage as well as cell cycle analysis. Induction of autophagy was indicated by increased acridine orange staining, RFP-LC3 redistribution, and detection of autophagic vesicles by electron microscopy, while autophagic flux was monitored based on p62 degradation. The autophagy inhibitors, chloroquine and bafilomycin A1, as well as genetic suppression of the autophagic signaling proteins Atg5 or Atg 7 attenuated the impact of the combination treatment of 1,25 D3 with radiation. In contrast to autophagy mediating the effects of the combination treatment, the autophagy induced by radiation alone was apparently cytoprotective in that either pharmacological or genetic inhibition increased sensitivity to radiation. These studies support the potential utility of vitamin D for improving the impact of radiation for breast cancer therapy, support the feasibility of combining chloroquine with radiation for the treatment of breast cancer, and demonstrate the existence of an "autophagic switch" from cytoprotective autophagy with radiation alone to cytotoxic autophagy with the 1,25 D3-radiation combination.     

Endoplasmic reticulum stress and cell death in mTORC1‐overactive cells is induced by nelfinavir and?enhanced by chloroquine

Inappropriate activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is common in cancer and has many cellular consequences including elevated endoplasmic reticulum (ER) stress. Cells employ autophagy as a critical compensatory survival mechanism during ER stress. This study utilised drug‐induced ER stress through nelfinavir in order to examine ER stress tolerance in cell lines with hyper‐active mTORC1 signalling. Our initial findings in wild type cells showed nelfinavir inhibited mTORC1 signalling and upregulated autophagy, as determined by decreased rpS6 and S6K1 phosphorylation, and SQTSM1 protein expression, respectively. Contrastingly, cells with hyper‐active mTORC1 displayed basally elevated levels of ER stress which was greatly exaggerated following nelfinavir treatment, seen through increased CHOP mRNA and XBP1 splicing. To further enhance the effects of nelfinavir, we introduced chloroquine as an autophagy inhibitor. Combination of nelfinavir and chloroquine significantly increased ER stress and caused selective cell death in multiple cell line models with hyper‐active mTORC1, whilst control cells with normalised mTORC1 signalling tolerated treatment. By comparing chloroquine to other autophagy inhibitors, we uncovered that selective toxicity invoked by chloroquine was independent of autophagy inhibition yet entrapment of chloroquine to acidified lysosomal/endosomal compartments was necessary for cytotoxicity. Our research demonstrates that combination of nelfinavir and chloroquine has therapeutic potential for treatment of mTORC1‐driven tumours.     

Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.     

Increased local antitumor effects of interleukin 2 liposomes in mice with MCA-106 sarcoma pulmonary metastases

The effects of liposome formulations of interleukin 2 (IL-2) and local route were studied in C57BL/6 mice with MCA-106 sarcoma pulmonary metastases. IL-2 liposomes made by hydration of powdered dimyristoyl-phosphatidylcholine with aqueous recombinant IL-2 had 95% of the IL-2 associated with the lipid fraction. When mice with pulmonary micrometastases were treated once daily with free cytokine on days 5, 6, and 7 after tumor inoculation, the intrathoracic route was superior to the i.p. or s.c. routes. When IL-2 liposomes were administered by the local intrathoracic route, significantly better antitumor effects (P less than 0.01) were seen compared to empty liposomes or free IL-2 as determined by (a) increased survival and (b) reduced numbers of pulmonary metastases. Minimal toxicity was observed. Results indicate that local route and incorporation of IL-2 in liposomes may enhance therapeutic efficacy and facilitate more practical daily dosing regimens.     

Pleotropic role of RNA binding protein CELF2 in autophagy induction

We previously reported that ionizing radiation (IR) mediates cell death through the induction of CUGBP elav‐like family member 2 (CELF2), a tumor suppressor. CELF2 is an RNA binding protein that modulates mRNA stability and translation. Since IR induces autophagy, we hypothesized that CELF2 regulates autophagy‐mediated colorectal cancer (CRC) cell death. For clinical relevance, we determined CELF2 levels in The Cancer Genome Atlas (TCGA). Role of CELF2 in radiation response was carried out in CRC cell lines by immunoblotting, immunofluorescence, autophagic vacuole analyses, RNA stability assay, quantitative polymerase chain reaction and electron microscopy. In vivo studies were performed in a xenograft tumor model. TCGA analyses demonstrated that compared to normal tissue, CELF2 is expressed at significantly lower levels in CRC, and is associated with better overall 5‐year survival in patients receiving radiation. Mechanistically, CELF2 increased levels of critical components of the autophagy cascade including Beclin‐1, ATG5, and ATG12 by modulating mRNA stability. CELF2 also increased autophagic flux in CRC. IR significantly induced autophagy in CRC which correlates with increased levels of CELF2 and autophagy associated proteins. Silencing CELF2 with siRNA, mitigated IR induced autophagy. Moreover, knockdown of CELF2 in vivo conferred tumor resistance to IR. These studies elucidate an unrecognized role for CELF2 in inducing autophagy and potentiating the effects of radiotherapy in CRC.     

氯喹促进顺铂诱导胃癌SGC7901细胞凋亡及其机制

  目的  研究自噬对顺铂（cisplatin,DDP）诱导的胃癌SGC7901细胞凋亡的影响,并初步探讨其可能机制。  方法  DDP和（或）氯喹处理胃癌SGC7901细胞,MTT法检测细胞增殖,流式细胞术检测细胞凋亡,MDC染色后荧光显微镜观察自噬囊泡,West. ern Blot检测自噬和凋亡相关蛋白。  结果  5 mg/L的顺铂作用于胃癌SGC7901细胞24 h,细胞凋亡率为21.07%±2.12%,同时观测到自噬囊泡增多和LC3-Ⅱ蛋白表达升高;氯喹特异性抑制自噬活性后,提高了顺铂诱导的细胞凋亡率（30.16%±3.54%,P < 0.05）;检测到凋亡相关蛋白Caspase-3和P53表达增加,Bcl-2蛋白表达下降。  结论  自噬在顺铂诱导胃癌SGC7901细胞凋亡的过程中起保护作用,氯喹抑制自噬后,可能通过激活P53蛋白及灭活Bcl-2蛋白的表达来促进细胞凋亡,联合应用顺铂和氯喹有望成为胃癌治疗的新策略。      

Polyphenol-rich extract of Pimenta dioica berries (Allspice) kills breast cancer cells by autophagy and delays growth of triple negative breast cancer in athymic mice

Bioactive compounds from edible plants have limited efficacy in treating advanced cancers, but they have potential to increase the efficacy of chemotherapy drugs in a combined treatment. An aqueous extract of berries of Pimenta dioica (Allspice) shows promise as one such candidate for combination therapy or chemoprevention. An aqueous extract of Allspice (AAE) was tested against human breast cancer (BrCa) cells in vitro and in vivo. AAE reduced the viability and clonogenic growth of several types of BrCa cells (IC₅₀ ≤ 100 μg/ml) with limited toxicity in non-tumorigenic, quiescent cells (IC₅₀ >200 μg/ml). AAE induced cytotoxicity in BrCa was inconsistent with apoptosis, but was associated with increased levels of autophagy markers LC3B and LC3B-positive puncta. Silencing the expression of autophagy related genes (ATGs) prevented AAE-induced cell death. Further, AAE caused inhibition of Akt/mTOR signaling, and showed enhanced cytotoxicity when combined with rapamycin, a chemotherapy drug and an inhibitor of mTOR signaling. Oral administration (gavage) of AAE into athymic mice implanted with MDA-MB231 tumors inhibited tumor growth slightly but not significantly (mean decrease ~ 14%, p ≥ 0.20) if mice were gavaged post-tumor implant. Tumor growth showed a significant delay (38%) in tumor palpability and growth rate (time to reach tumor volume ≥ 1,000 mm³) when mice were pre-dosed with AAE for two weeks. Analysis of tumor tissues showed increased levels of LC3B in AAE treated tumors, indicating elevated autophagic tumor cell death in vivo in treated mice. These results demonstrate antitumor and chemo-preventive activity of AAE against BrCa and potential for adjuvant to mTOR inhibition.     

Augmentation of NVP-BEZ235's anticancer activity against human lung cancer cells by blockage of autophagy

Autophagy is a cellular lysosomal degradation pathway essential for regulation of cell survival and death to maintain homeostasis. This process is negatively regulated by mammalian target of rapamycin (mTOR) signaling and often counteracts efficacy of certain cancer therapeutic agents. NVP-BEZ235 (BEZ235) is a novel, orally bioavailable dual PI3K/mTOR inhibitor that has exhibited promising activity against non-small cell lung cancer (NSCLC) in preclinical models. The current study focuses on evaluating the role of BEZ235 in regulating autophagy. BEZ235 was effective in inhibiting the growth of NSCLC cells including induction of apoptosis. It also potently induced the expression of type-II LC3, indicating induction of autophagy. When BEZ235 was used in combination with the lysosomal or autophagic inhibitor chloroquine (CQ), enhanced inhibitory effects on monolayer growth and colony formation of NSCLC cells was observed. In addition, enhanced induction of apoptosis was also detected in cells exposed to the combination of BEZ235 and CQ. Moreover, the combination of BEZ235 and CQ was more effective than each single agent alone in inhibiting the growth of NSCLC xenografts in nude mice. Thus, induction of autophagy by BEZ235 appears to be a survival mechanism that may counteract its anticancer effects. Based on these, we suggest a strategy to enhance BEZ235's anticancer efficacy by blockade of autophagy.     

Polysaccharides from sporoderm-removed spores of Ganoderma lucidum induce apoptosis in human gastric cancer cells via disruption of autophagic flux

The sporoderm-broken spores of Ganoderma lucidum (G. lucidum) polysaccharide (BSGLP) have been demonstrated to inhibit carcinogenesis in several types of cancer. However, to the best of our knowledge, the anticancer effects of polysaccharides extracted from the newly developed sporoderm-removed spores of G. lucidum (RSGLP) have not been assessed. The present study first compared the anticancer effects of RSGLP and BSGLP in three gastric cancer cell lines and it was found that RSGLP was more potent than BSGLP in decreasing gastric cancer cell viability. RSGLP significantly induced apoptosis in AGS cells, accompanied by downregulation of Bcl-2 and pro-caspase-3 expression levels, and upregulation of cleaved-PARP. Furthermore, RSGLP increased LC3-II and p62 expression, indicative of induction of autophagy and disruption of autophagic flux in AGS cells. These results were further verified by combined treatment of AGS cells with the late-stage autophagy inhibitor chloroquine, or early-stage autophagy inducer rapamycin. Adenoviral transfection with mRFP-GFP-LC3 further confirmed that autophagic flux was inhibited by RSGLP in AGS cells. Finally, the present study demonstrated that the RSGLP-induced autophagy and disruption of autophagic flux disruption was, at least in part, responsible for RSGLP-induced apoptosis in AGS cells. The results of the present study demonstrated for the first time that RSGLP is more effective than BSGLP in inhibiting gastric cancer cell viability, and RSGLP may serve as a promising autophagy inhibitor in the management of gastric cancer.     

Intratumoral injection of interleukin-2 augments the local and abscopal effects of radiotherapy in murine rectal cancer

Recent studies have suggested that tumor shrinkage in response to radiotherapy (RT) is greatly dependent on the host immune response. A Balb/c mouse model of simultaneous subcutaneous tumor and liver metastasis of Colon26 was prepared and, after irradiation of the subcutaneous tumor (2 Gy × 5 day × 2 cycles), interleukin-2 (IL-2) (2 × 10(4) U) was injected intra-tumorally, and the fate of both the subcutaneous tumor and liver metastatic lesions was evaluated. Intratumoral injection of IL-2 greatly enhanced the anti-tumor effects of RT and completely eradicated the established subcutaneous tumor. Interestingly, although RT was given locally to the subcutaneous tumor, liver metastasis formation was also inhibited in mice receiving only local RT. More impressively, the combination of RT + IL-2 completely inhibited liver metastasis formation. Splenocytes in mice receiving RT + IL-2 contained a higher percentage of CD4(+) T cells, but lower percentages of CD4(+)CD25(+) regulatory T cells and CD11b(+) Gr-1(+) myeloid-derived suppressor cells. Immunohistochemical investigation of human rectal cancer revealed that the density of CD8(+) cells infiltrating into irradiated rectal tumor was positively associated with a lower frequency of distant metastasis as well as histological response grade. Local administration of IL-2 not only enhances shrinkage of the irradiated tumor itself, but can also suppress the development of distant metastasis located outside the RT field, possibly though the induction of a systemic T cell response. Augmentation of T-cell-mediated antitumor immunity during RT might be critical for improvement of the clinical efficacy of neoadjuvant RT for the treatment of advanced rectal cancer.     

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.     

Inhibiting autophagy increases epirubicin's cytotoxicity in breast cancer cells

Chemotherapy, radiotherapy, and endocrinotherapy are documented to induce autophagy among breast cancer cells, but the role of autophagy in this disease has been attributed as cytoprotective as well as tumor‐suppressing. Thus we studied MDA‐MB‐231 and SK‐BR‐3 breast cancer cell lines treated with epirubicin (EPI) to assess autophagy and apoptosis. We found out that EPI induced apoptosis and autophagy in both cell lines. The lysosomal inhibitor bafilomycin A1 inhibited cellular autophagy and enhanced EPI‐triggered apoptosis, perhaps due to inhibition of autolysosome formation, which then inhibited autophagic effects of engulfing and clearing damaged mitochondria. This inhibition increased mitochondrial cytochrome C release which augmented epirubicin‐induced caspase‐dependent apoptosis and cytotoxicity. In addition, the lysosomal neutralizing agent ammonia chloride (AC), and Atg7 knockdown by siRNA, could inhibit epirubicin‐triggered autophagy, enhance cytotoxicity, and increase caspase‐9‐ and caspase‐3‐dependent apoptosis. Thus, autophagy plays a prosurvival role in EPI‐treated MDA‐MB‐231 and SK‐BR‐3 cells, and autophagy inhibition can potentially reverse this effect and increase the cytotoxicity of EPI.     

人参皂甙Rg1协同诱生LAK细胞的抗瘤活性

淋巴因子激活性杀伤细胞(LAK细胞)过继输入疗法已经被证明是一种有效的抗肿瘤免疫治疗措施,但此疗法的临床应用却受到LSK细胞抗瘤活性尚低问题的困扰.我们曾经证明人参总皂甙能够增强LAK细胞杀伤新鲜急性白血病细胞的活性,本文进一步检测了人参总皂甙的单体之一人参皂甙Rg_1(Ginsenoside Rg_1)对LAK细胞抗瘤活性的影响,报告如下.     

Autophagy and autophagic cell death are next targets for elimination of the resistance to tyrosine kinase inhibitors

Autophagy, a cellular degradation system has been demonstrated in some hematopoietic malignant cell lines, but there is much still remaining to be known about its role and the mechanisms. We observed the excessive autophagy in chronic myelogenous leukemia (CML) cell line, K562, associated with treatment of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which can induce K562 cells to differentiate into megakaryocytic lineage. Confocal microscopic analysis demonstrated that autophagic cells did not express a megakaryocyte marker, the CD41 molecule, indicating that the autophagy was independent of megakaryocytic differentiation. After remarkable autophagic degradation, the cells finally underwent autophagic cell death (APCD). On the other hand, a block of TPA-induced autophagy by chloroquine rapidly promoted cell death that was not APCD. This result suggested that autophagy regulated two mechanisms in K562 cells: both the cell survival system and APCD. To confirm that autophagy regulates the cell survival system in K562 cells, imatinib was used to induce cell death in K562 cells. Autophagy has not been considered during imatinib treatment; nonetheless, co-treatment with imatinib and chloroquine markedly enhanced imatinib-induced cell death, compared to K562 cells treated only with imatinib. Furthermore, imatinib-resistant cell lines, BaF3/T315I and BaF3/E255K, also underwent cell death by co-treatment with imatinib and chloroquine. From these data, we concluded that autophagy is deeply related to the cell survival system and that inhibition of autophagy accelerates TPA- or imatinib-induced cell death. The block of autophagy could be a new strategy in the treatment of CML.     

Antagonistic effects of chloroquine on autophagy occurrence potentiate the anticancer effects of everolimus on renal cancer cells

Renal cell carcinoma is an aggressive disease often asymptomatic and weakly chemo-radiosensitive. Currently, new biologic drugs are used among which everolimus, an mTOR inhibitor, that has been approved for second-line therapy. Since mTOR is involved in the control of autophagy, its antitumor capacity is often limited. In this view, chloroquine, a 4-alkylamino substituted quinoline family member, is an autophagy inhibitor that blocks the fusion of autophagosomes and lysosomes. In the present study, we evaluated the effects of everolimus alone or in combination with chloroquine on renal cancer cell viability and verified possible synergism. Our results demonstrate that renal cancer cells are differently sensitive to everolimus and chloroquine and the pharmacological combination everolimus/chloroquine was strongly synergistic inducing cell viability inhibition. In details, the pharmacological synergism occurs when chloroquine is administered before everolimus. In addition, we found a flow autophagic block and shift of death mechanisms to apoptosis. This event was associated with decrease of Beclin-1/Bcl^-2 complex and parallel reduction of anti-apoptotic protein Bcl^-2 in combined treatment. At last, we found that the enhancement of apoptosis induced by drug combination occurs through the intrinsic mitochondrial apoptotic pathway activation, while the extrinsic pathway is involved only partly following its activation by chloroquine. These results provide the basis for new therapeutic strategies for the treatment of renal cell carcinoma after appropriate clinical trial.