MiR-153-5p promotes sensibility of colorectal cancer cells to oxaliplatin via targeting Bcl-2-mediated autophagy pathway

ABSTRACT

Oxaliplatin (L-OHP) is one of the effective chemotherapeutic drugs for colorectal cancer (CRC). Further investigation into the molecular mechanism of chemoresistance could improve outcomes for patients with colorectal cancer. Recently, microRNAs have been reported as a key in drug resistance of tumors. In this study, we aimed to investigate the effects of miR-153-5p in L-OHP-resistant CRC cells, and its underlying mechanism. Downregulation of miR-153-5p was observed in CRC cells, while upregulation of miR-153-5p enhances the chemosensitivity of CRC/L-OHP cells. The autophagy of CRC/L-OHP cells was markedly increased after exposure to L-OHP but abolished by the upregulation of miR-153-5p. Dual-luciferase reporter assays validated that Bcl-2 was a direct target of miR-153-5p. In conclusion, our data suggested that miR-153-5p was a mediator of cisplatin resistance in colorectal cancer by affecting Bcl-2-mediated autophagy, indicating a new therapeutic target for CRC treatment.     

PEG-liposomal oxaliplatin induces apoptosis in human colorectal cancer cells via Fas/FasL and caspase-8

Since cellular uptake of PEG [poly(ethylene glycol)]-liposomal L-OHP (oxaliplatin) induces bioactive changes in CRC (colorectal cancer), we have investigated its apoptotic effect and anticancer mechanism. Human CRC SW480 cells were treated with PEG-liposomal L-OHP and a caspase-8 inhibitor [Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-dl-Asp-fluoromethylketone)]. Apoptosis was measured by FCM (flow cytometry) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay. Expression of Fas/FasL and cytochrome c was detected using FCM and an immunofluorescence assay. Expression of caspase-8, Bid, caspase-9, caspase-7 and activated caspase-3 (P17) was examined by Western blot analyses. The results indicated that PEG-liposomal L-OHP (28 μg/ml L-OHP) induced marked apoptosis in SW480 cells compared with 28 μg/ml free L-OHP. The expression levels of Fas, FasL, cytochrome c, caspase-9, caspase-7 and activated caspase-3 proteins were up-regulated, with a corresponding increase in apoptosis; however, expression of caspase-8 and Bid were down-regulated as apoptosis increased. When cells were treated with Z-IETD-FMK, apoptosis was inhibited, but there was little impact on the expression of Fas, FasL, cytochrome c, Bid, caspase-9, caspase-7 and activated caspase-3. These findings indicate that PEG-liposomal L-OHP enhances the anticancer potency of the chemotherapeutic agent; moreover, Fas/FasL and caspase-8 signalling pathways play a key role in mediating PEG-liposomal L-OHP-induced apoptosis.     

miR-129 promotes apoptosis and enhances chemosensitivity to 5-fluorouracil in colorectal cancer

Resistance to fluoropyrimidine-based chemotherapy is the major reason for the failure of advanced colorectal cancer (CRC) treatment. The lack of ability of tumor cells to undergo apoptosis after genotoxic stress is the key contributor to this intrinsic mechanism. Mounting evidence has demonstrated that non-coding microRNAs (miRNAs) are crucial regulators of gene expression, in particular, under acute genotoxic stress. However, there is still limited knowledge about the role of miRNAs in apoptosis. In this study, we discovered a novel mechanism mediated by microRNA-129 (miR-129) to trigger apoptosis by suppressing a key anti-apoptotic protein, B-cell lymphoma 2 (BCL2). Ectopic expression of miR-129 promoted apoptosis, inhibited cell proliferation and caused cell-cycle arrest in CRC cells. The intrinsic apoptotic pathway triggered by miR-129 was activated by cleavage of caspase-9 and caspase-3. The expression of miR-129 was significantly downregulated in CRC tissue specimens compared with the paired normal control samples. More importantly, we demonstrated that miR-129 enhanced the cytotoxic effect of 5-fluorouracil both in vitro and in vivo. These results suggest that miR-129 has a unique potential as a tumor suppressor and a novel candidate for developing miR-129-based therapeutic strategies in CRC.     

Ursolic acid synergistically enhances the therapeutic effects of oxaliplatin in colorectal cancer

Oxaliplatin is a key drug in chemotherapy of colorectal cancer (CRC). However, its efficacy is unsatisfied due to drug resistance of cancer cells. In this study, we tested whether a natural agent, ursolic acid, was able to enhance the efficacy of oxaliplatin for CRC. Four CRC cell lines including SW480, SW620, LoVo, and RKO were used as in vitro models, and a SW620 xenograft mouse model was used in further in vivo study. We found that ursolic acid inhibited proliferation and induced apoptosis of all four cells and enhanced the cytotoxicity of oxaliplatin. This effect was associated with down-regulation of Bcl-xL, Bcl-2, survivin, activation of caspase-3, 8, 9, and inhibition of KRAS expression and BRAF, MEK1/2, ERK1/2, p-38, JNK, AKT, IKKα, IκBα, and p65 phosphorylation of the MAPK, PI3K/AKT, and NF-κB signaling pathways. The two agents also showed synergistic effects against tumor growth in vivo. In addition, ursolic acid restored liver function and body weight of the mice treated with oxaliplatin. Thus, we concluded that ursolic acid could enhance the therapeutic effects of oxaliplatin against CRC both in vitro and in vivo, which offers an effective strategy to minimize the burden of oxaliplatin-induced adverse events and provides the groundwork for a new clinical strategy to treat CRC.     

ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

Autophagy critically contributes to metabolic reprogramming and chromosomal stability. It has been reported that monoallelic loss of the essential autophagy gene BECN1 (encoding BECN1/Beclin 1) promotes cancer development and progression. However, the mechanism by which BECN1 is inactivated in malignancy remains largely elusive. We have previously reported a tumor suppressor role of ABHD5 (abhydrolase domain containing 5), a co-activator of PNPLA2 (patatin like phospholipase domain containing 2) in colorectal carcinoma (CRC). Here we report a noncanonical role of ABHD5 in regulating autophagy and CRC tumorigenesis. ABHD5 directly competes with CASP3 for binding to the cleavage sites of BECN1, and consequently prevents BECN1 from being cleaved by CASP3. ABHD5 deficiency provides CASP3 an advantage to cleave and inactivate BECN1, thus impairing BECN1-induced autophagic flux and augmenting genomic instability, which subsequently promotes tumorigenesis. Notably, clinical data also confirm that ABHD5 proficiency is significantly correlated with the expression levels of BECN1, LC3-II and CASP3 in human CRC tissues. Our findings suggest that ABHD5 possesses a PNPLA2-independent function in regulating autophagy and tumorigenesis, further establishing the tumor suppressor role of ABHD5, and offering an opportunity to develop new approaches aimed at preventing CRC carcinogenesis.     

Circ-ERBB2 knockdown sensitized colorectal cancer cells to 5-FU via miR-181a-5p/PTEN/Akt pathway

Colorectal cancer (CRC) is the fourth most deadly cancer worldwide, drug resistance impedes treatment of CRC. It is still urgent to find new molecular targets to improve the sensitivity of chemotherapeutic drugs. In this study, circ-ERBB2 was upregulated in CRC cells. Upregulation of circ-ERBB2 promoted CRC cells proliferation and clone formation, but inhibited apoptosis. We identified miR-181a-5p as circ-ERBB2's target. The effect of miR-181a-5p on CRC cells was contrary to circ-ERBB2, miR-181a-5p downregulation abolished the function of circ-ERBB2 silencing in CRC cells. In addition, phosphatase and tensin homolog (PTEN) was verified as miR-181a-5p's downstream target, circ-ERBB2 activates the Akt pathway and inhibits cell apoptosis through modulating miR-181a-5p/PTEN. Circ-ERBB2 silencing significantly reduced CRC cell resistance to 5-FU. miR-181a-5p downregulation abolished the role of circ-ERBB2 knockdown in CRC cell resistance to 5-FU. In conclusion, upregulation of circ-ERBB2 promoted the malignancy of CRC and reduced CRC cell resistance to 5-FU. Besides, additional mechanism study provided a novel regulatory pathways that circ-ERBB2 knockdown promoted CRC cell sensitivity to 5-FU by regulating miR-181a-5p/PTEN/Akt pathway. This research indicated that circ-ERBB2 may be a valuable biomarker for the diagnosis and treatment of CRC.     

Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.     

Inhibition of KHSRP sensitizes colorectal cancer to 5-fluoruracil through miR-501-5p-mediated ERRFI1 mRNA degradation

K-homology (KH)-type splicing regulatory protein (KHSRP) is an RNA binding protein that participates in RNA variable splicing and stability, and facilitates the biogenesis of miRNAs that target mRNA. However, to date, the role of KHSRP in colorectal cancer (CRC) progression has not been reported. In this study, the function of KHSRP in CRC proliferation and 5-fluoruracil (5-FU) resistance was investigated. The upregulation of KHSRP expression was confirmed in CRC patient tissues and two CRC cell lines. Manipulating KHSRP expression altered cell proliferation and 5-FU resistance in CRC cells. ERRFI1, a downstream effector of KHSRP in CRC cells, reduced CRC cell proliferation. Sensitivity to 5-FU mediated by KHSRP knockdown was reversed by ERRFI1 knockdown. We found that KHSRP decreased ERRFI1 mRNA expression indirectly. By screening KHSRP-regulated miRNAs, we further found that miR-501-5p directly combines with KHSRP in CRC cells. Mechanistically, the results of a luciferase assay suggested that miR-501-5p directly binds to the ERRFI1 3′-untranslated region. Taken together, our data indicated that modification of ERRFI1 by KHSRP occurs through miR-501-5p, an essential mechanism driving CRC proliferation and 5-FU resistance. Insight into this mechanism may provide novel targets for overcoming drug resistance in CRC.     

Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

Colorectal cancer (CRC) is the most common digestive cancer in the Western world. Despite effective therapies, resistance and/or recurrence frequently occur. The present study investigated the impact of two survival pathways—neurotrophic factors (TrkB/BDNF) and autophagy—on cell fate and tumour evolution. In vitro studies were performed on two CRC cell lines, SW480 (primary tumour) and SW620 (lymph node invasion), which were also used for subcutaneous xenografts on a nude mouse model. In addition, the presence of neurotrophic factors (NTs) and autophagy markers were assessed in tissue samples representative of different stages. On the basis of our previous study (which demonstrated that TrkB overexpression is associated with prosurvival signaling in CRC cells), we pharmacologically inhibited NTs pathways with K252a. As expected, an inactivation of the PI3K/AKT pathway was observed and CRC cells initiated autophagy. Conversely, blocking the autophagic flux with chloroquine or with ATG5‐siRNA overactivated TrkB/BDNF signaling. In vitro, dual inhibition improved the effectiveness of single treatment by significantly reducing metabolic activity and enhancing apoptotic cell death. These findings were accentuated in vivo, in which dual inhibition induced a spectacular reduction in tumour volume following long‐term treatment (21 days for K252a and 12 days for CQ). Finally, significant amounts of phospho‐TrkB and LC3II were found in the patients’ tissues, highlighting their relevance in CRC tumour biology. Taken together, our results show that targeting NTs and autophagy pathways potentially constitutes a new therapeutic approach for CRC.     

Annexin A3 depletion overcomes resistance to oxaliplatin in colorectal cancer via the MAPK signaling pathway

Colorectal cancer (CRC) is a common disease with high mortality and morbidity. Annexin A3 (ANXA3) belongs to the structurally homologous family of Ca²⁺ and phospholipid-binding proteins. This study aimed to investigate the effects and potential mechanisms of ANXA3 on oxaliplatin (Ox) resistance in CRC. We generated two human CRC cell lines (HCT116/Ox and SW480/Ox) with acquired Ox resistance and determined their resistance properties. ANXA3 expression and cell apoptosis, migration and invasion also were evaluated. We found that cell viability of HCT116/Ox and SW480/Ox was higher than that in parental cells in the presence of Ox. ANXA3 was highly expressed in HCT116/Ox and SW480/Ox cells. ANXA3 downregulation diminished cell survival, migration and invasion, while increased the apoptosis of HCT116 and SW480 with or without Ox. Moreover, depletion of ANXA3 reduced cell viability and BrdU incorporation, increased cell apoptosis and c-caspase 3 expression in HCT116/Ox with or without Ox. A transwell assay determined that knockdown of ANXA3 impeded the migration and invasion of HCT116/Ox and SW480/Ox cells. Additionally, phosphorylation of extracellular signal–regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) decreased upon ANXA3 depletion in HCT116/Ox cells, and ANXA3 silencing suppressed Ox-induced activation of ERK and JNK signaling pathway. ANXA3 downregulation reduced Ox resistance in CRC, and treatment with the ERK inhibitor PD098059 or JNK inhibitor SP600125 contributed to this process. These results indicate that silencing ANXA3 could overcome Ox resistance in CRC via the mitogen-activated protein kinase signaling pathway.     

Suppression of TWIST1 enhances the sensitivity of colon cancer cells to 5-fluorouracil

The 5-fluorouracil (5FU)-based adjuvant chemotherapy improves the survival of patients with colorectal cancer, however the main obstacle affecting its effectiveness is a drug resistance. Our study aimed to investigate the impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU. The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-shTWIST1). The suppression of TWIST1 expression induced changes in the expression pattern of epithelial to mesenchymal transition markers, reduced the cells proliferation rate, increased their sensitivity to serum withdrawn, and increased the cytotoxic effect of 5FU. However, significantly higher 5FU cytotoxicity was observed in HT29 cell cultures. Cells with silenced TWIST1 displayed altered expression of enzymes metabolizing 5FU. The expression level of dihydropyrimidine dehydrogenase, and thymidylate synthase decreased significantly in HT29 shTWIST1 cells, but not in HCT116 shTWIST1 cells. On the other hand, significant increases in the expression levels of thymidine phosphorylase, and uridine phosphorylase 1 were seen in both cell lines with suppressed expression of TWIST1. The changes in enzymes expression were mirrored by enzymatic activities. In conclusion, our observations point to TWIST1 as a target protein to enhance the sensitivity of colorectal cancer cells to 5FU.     

Retracted: Long noncoding RNA HAGLROS regulates apoptosis and autophagy in colorectal cancer cells via sponging miR-100 to target ATG5 expression

The aim of this study was to explore the relationship between the expression of HOXD antisense growth-associated long noncoding RNA (HAGLROS) and prognosis of patients with colorectal cancer (CRC), as well as the roles and regulatory mechanism of HAGLROS in CRC development. The HAGLROS expression in CRC tissues and cells was detected. The correlation between HAGLROS expression and survival time of CRC patients was investigated. Moreover, HAGLROS was overexpressed and suppressed in HCT-116 cells, followed by detection of cell viability, apoptosis, and the expression of apoptosis-related proteins and autophagy markers. Furthermore, the association between HAGLROS and miR-100 and the potential targets of miR-100 were investigated. Besides, the regulatory relationship between HAGLROS and PI3K/AKT/mTOR pathway was elucidated. The results showed that HAGLROS was highly expressed in CRC tissues and cells. Highly expression of HAGLROS correlated with a shorter survival time of CRC patients. Moreover, knockdown of HAGLROS in HCT-116 cells induced apoptosis by increasing the expression of Bax/Bcl-2 ratio, cleaved-caspase-3, and cleaved-caspase-9, and inhibited autophagy by decreasing the expression of LC3II/LC3I and Beclin-1 and increasing P62 expression. Furthermore, HAGLROS negatively regulated the expression of miR-100, and HAGLROS controlled HCT-116 cell apoptosis and autophagy through negatively regulation of miR-100. Autophagy related 5 (ATG5) was verified as a functional target of miR-100 and miR-100 regulated HCT-116 cell apoptosis and autophagy through targeting ATG5. Besides, HAGLROS overexpression activated phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. In conclusion, a highly expression of HAGLROS correlated with shorter survival time of CRC patients. Downregulation of HAGLROS may induce apoptosis and inhibit autophagy in CRC cells by regulation of miR-100/ATG5 axis and PI3K/AKT/mTOR pathway.     

MARCH5 RNA promotes autophagy,migration, and invasion of ovarian cancer cells

MARCH5 is a crucial regulator of mitochondrial fission. However, the expression and function of MARCH5 in ovarian cancer have not been determined. This study investigated the expression and function of MARCH5 in ovarian cancer with respect to its potential role in the tumorigenesis of the disease as well as its usefulness as an early diagnostic marker. We found that the expression of MARCH5 was substantially upregulated in ovarian cancer tissue in comparison with the normal control. Silencing MARCH5 in SKOV3 cells decreased TGFB1-induced cell macroautophagy/autophagy, migration, and invasion in vitro and in vivo, whereas the ectopic expression of MARCH5 in A2780 cells had the opposite effect. Mechanistic investigations revealed that MARCH5 RNA may function as a competing endogenous RNA (ceRNA) to regulate the expression of SMAD2 and ATG5 by competing for MIR30A. Knocking down SMAD2 or ATG5 can block the effect of MARCH5 in A2780 cells. Also, silencing the expression of MARCH5 in SKOV3 cells can inhibit the TGFB1-SMAD2/3 pathway. In contrast, the ectopic expression of MARCH5 in A2780 cells can activate the TGFB1-SMAD2/3 pathway. In turn, the TGFB1-SMAD2/3 pathway can regulate MARCH5 and ATG5 through MIR30A. Overall, the results of this study identified MARCH5 as a candidate oncogene in ovarian cancer and a potential target for ovarian cancer therapy.     

Energy deprivation by silibinin in colorectal cancer cells

Small molecules with the potential to initiate different types of programmed cell death could be useful ‘adjunct therapy’ where current anticancer modalities fail to generate significant activity due to a defective apoptotic machinery or resistance of cancer cells to the specific death mechanism induced by that treatment. The current study identified silibinin, for the first time, as one such natural agent, having dual efficacy against colorectal cancer (CRC) cells. First, silibinin rapidly induced oxidative stress in CRC SW480 cells due to reactive oxygen species (ROS) generation with a concomitant dissipation of mitchondrial potential (ΔΨ_m) and cytochrome c release leading to mild apoptosis as a biological effect. However, with increased exposure to silibinin, cytoplasmic vacuolization intensified within the cells followed by sequestration of the organelles, which inhibits the further release of cytochrome c. Interestingly, this decrease in apoptotic response correlated with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Mechanistic studies revealed that silibinin strongly inhibited PIK3CA-AKT–MTOR but activated MAP2K1/2-MAPK1/3 pathways for its biological effects. Corroborating these effects, endoplasmic reticulum stress was generated and glucose uptake inhibition as well as energy restriction were induced by silibinin, thus, mimicking starvation-like conditions. Further, the cellular damage to tumor cells by silibinin was severe and irreparable due to sustained interference in essential cellular processes such as mitochondrial metabolism, phospholipid and protein synthesis, suggesting that silibinin harbors a deadly ‘double-edged sword’ against CRC cells thereby further advocating its clinical effectiveness against this malignancy.     

The role of microRNAs in 5-FU resistance of colorectal cancer: Possible mechanisms

Colorectal cancer (CRC) is one of the most common cancers globally. Despite recent advances in therapeutic approaches, this cancer continues to have a poor prognosis, particularly when diagnosed late. 5-Fluorouracil (5-FU) has been commonly prescribed for patients with CRC, but resistance to 5-FU is one of the main reasons for failure in the treatment of this condition. Recently, microRNAs (miRNAs) have been established as a means of modifying the signaling pathways involved in initiation and progression of CRC and their role as oncogene or tumor suppressor have been investigated in various studies. Moreover, miRNAs through various mechanisms play an important role in inducing tumor resistance or sensitivity to anticancer drugs. Detecting and targeting these mechanisms may be a new therapeutic approach. This review summarizes the current knowledge about the potential roles of miRNAs in 5-FU resistance, with particular emphasis on molecular mechanism involved.     

IRX5 prompts genomic instability in colorectal cancer cells

The Iroquois homeobox gene 5 (IRX5), one of the members of the Iroquois homeobox family, has been identified to correlate with worse prognosis in many cancers, including colorectal cancer (CRC). In this study, upregulation of IRX5 revealed a great reduction in the proliferation of CRC colorectal cancer cell line SW480 and DLD-1, which was accompanied by G1/S arrest, increased expression in cyclin E1, P21, and P53 and a decrease in cyclin A2, B1, and D1. Furthermore, IRX5-mediated an increase expression of RH2A protein, the biomarker of DNA damage. Consequently, the SA-β-gal level is higher in IRX5-overexpression cells compared to control ones, which showed elevated DNA damage triggered cellular senescence. Recapitulating the above findings, IRX5 exhibited higher levels of genomic instability. IRX5 may be a perspective target for cancer therapy and it deserves further investigation.