Doxorubicin is conventionally used in chemotherapy against hepatocellular carcinoma (HCC), but acquired resistance developed during long-term therapy limits its benefits. Autophagy, a conserved catabolic process for cellular self-protection and adaptation to the changing environment, is regarded as a potential clinical target to overcome doxorubicin resistance. In this study, the potential role of miR-223 in modulating doxorubicin-induced autophagy and sensitivity were evaluated in four transfected human HCC cell lines, and the in vivo relevance was assessed using a mouse xenograft model of HCC. We found that the well-defined miR-223 is expressed at low levels in doxorubicin treated HCC cells and that miR-223 overexpression inhibits the doxorubicin-induced autophagy that contributes to chemoresistance. Blockade of autophagic flux by chloroquine resulted in the failure of miR-223 inhibitor to suppress doxorubicin sensitivity of HCC cells. We further identified FOXO3a as a direct downstream target of miR-223 and primary mediator of the regulatory effect of miR-223 on doxorubicin-induced autophagy and chemoresistance in HCC cells. Finally, we confirmed the enhancement of doxorubicin sensitivity by agomiR-223 in xenograft models of HCC. These findings establish a novel miRNA-based approach for autophagy interference to reverse doxorubicin resistance in future chemotherapy regimens against human HCC.

Lung cancer is the leading cause of cancer death worldwide. Recently, two plant-derived drugs triptolide (TP) and hydroxycamptothecin (HCPT) both have shown broad-spectrum anticancer activities. Our previous study documented that combination treatment with these two drugs acted more effectively than mono-therapy, however, the molecular basis underlying the synergistic cytotoxicity remains poorly understood. In this study, we aimed to clarify the molecular mechanism of TP/HCPT anticancer effect in A549 lung adenocarcinoma cells, by investigating the involvement of phosphatase 2A (PP2A) and PP2A-regulated mitogen-activated protein kinases (MAPKs) and Akt signaling pathways. The results showed that TP and HCPT synergistically exerted cytotoxicity in the growth of A549 cells. Combinatorial TP/HCPT treatment significantly enhanced the activation of caspase-3 and -9, Bax/Bcl-2 ratio, release of cytochrome c from mitochondrial and subsequent apoptosis. While the Akt survival pathway was inhibited, ERK and p38 MAPKs were dramatically activated. Furthermore, the activity of PP2A was significantly augmented. Regulation of p38, ERK and Akt by PP2A was demonstrated, by using a specific PP2A inhibitor okadaic acid (OA). Finally, pharmacological inhibitors OA, SB203580, SP600125 and PD98059 confirm the role of PP2A and its substrates ERK, p38 MAPK and Akt in mediating TP/HCPT-induced apoptosis. Taken together, this study provides the first evidence for a synergistic TP/HCPT anti-cancer activity in A549 cells and also supports a critical role of PP2A and PP2A-regulated signaling pathways, providing new insight into the mode of action of TP/HCPT in cancer therapy.

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Background: Cisplatin is widely used as a first-line treatment for non-small cell lung cancer (NSCLC), but chemoresistance remains a major clinical obstacle for efficient use. As a microRNA, miR-223 was reported to promote the doxorubicin resistance of NSCLC. However, whether miR-223 is also involved in cisplatin resistance of NSCLC and the mechanism miR-223 involved in drug resistance is unclear. Accumulated evidence has shown that abnormal autophagy is associated with tumor chemoresistance. The study aimed to study the role of miR-223 on cisplatin sensitivity in NSCLC and uncover the potential mechanisms. Methods: NSCLC cells transfected with mimic or inhibitor for miR-223 was assayed for chemoresistance in vitro. MiR-223 expression was assessed by quantitative real-time PCR (qRT-PCR). Western blot were used to study the expression level of F-box/WD repeat-containing protein 7 (FBXW7) and autophagy-related protein. The effect of miR-223 on cisplatin sensitivity was examined by using CCK-8, EdU assays and Autophagic flux assay. Luciferase assays, EdU assays and small interfering RNA were performed to identify the targets of miR-223 and the mechanism by which it promotes treatment resistance. Xenograft models were established to investigate the effect of mir-223 on cisplatin sensitivity. Results: In the present study, we found that the level of miR-223 was significantly positively correlated with cisplatin resistance. MiR-223 overexpression made NSCLC cells resistant to cisplatin treatment. We further found that autophagy mediated miR-223-mediated cisplatin resistance in NSCLC cells. Further mechanistic research demonstrated that miR-223 directly targeted FBXW7. The overexpression of miR-223 could inhibit the level of FBXW7 protein expression, thus promoting autophagy and making NSCLC cells resistant to cisplatin. Finally, we confirmed the increased effect of cisplatin sensitivity by miR-223 Antagomir in xenograft models of NSCLC. Conclusions: Our results demonstrate that miR-223 could enhance autophagy by targeting FBXW7 in NSCLC cells. Inhibition of autophagy by miR-223 knockdown provides a novel treatment strategy to alleviate cisplatin resistance in NSCLC.

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IntroductionMicroRNAs function as oncogenes or tumor suppressors in the development of various human cancers. We investigated the effect of microRNA-145 (miR-145) on colorectal cancer (CRC) cell invasion and migration.MethodsThe levels of miR-145 in CRC cells were examined by quantitative PCR; Western blotting was used to detect TWIST1 (twist family bHLH transcription factor 1) protein and the epithelial–mesenchymal transition (EMT)-related proteins (E-cadherin, vimentin). Then, we transfected miR-145 mimics or inhibitor into CRC cells and used the wound healing and Transwell invasion assays to investigate their migration and invasive capability, respectively.ResultsThe miR-145 mimics suppressed CRC cell invasion and migration significantly; in contrast, miR-145 downregulation had the opposite effect. Furthermore, miR-145 regulated TWIST1 levels negatively at transcriptional level. TWIST1 knockdown significantly inhibited the CRC cell migration ability and the number of CRC cells that crossed the Transwell membrane. There was no significant difference in terms of migration and invasive capability after the cells had been transfected with miR-145 mimics or inhibitor plus TWIST1 small interfering RNA (siRNA) as compared to the TWIST1 siRNA–only group. Furthermore, we demonstrate that the inhibition of miR-145 could enhance the capability for lung metastasis in vivo.ConclusionTaken together, these findings indicate that miR-145 acts as a new tumor suppressor by regulating TWIST1 and plays a vital role in the invasive and migration ability of CRC cells.

Background: Clear cell renal cell carcinoma (ccRCC) is one of the most prevalent and invasive histological subtypes among all renal cell carcinomas (RCC). Cancer cell metabolism, particularly glucose metabolism, has been reported as a hallmark of cancer. However, the characteristics of glucose metabolism-related gene sets in ccRCC have not been systematically profiled. Methods: In this study, we downloaded a gene expression profile and glucose metabolism-related gene set from TCGA (The Cancer Genome Altas) and MSigDB, respectively, to analyze the characteristics of glucose metabolism-related gene sets in ccRCC. We used a multivariable Cox regression analysis to develop a risk signature, which divided patients into low- and high- risk groups. In addition, a nomogram that combined the risk signature and clinical characteristics was created for predicting the 3- and 5-year overall survival (OS) of ccRCC. The accuracy of the nomogram prediction was evaluated using the area under the receiver operating characteristic curve (AUC) and a calibration plot. Results: A total of 231 glucose metabolism-related genes were found, and 68 differentially expressed genes (DEGs) were identified. After screening by univariate regression analysis, LASSO regression analysis and multivariable Cox regression analysis, six glucose metabolism-related DEGs ( FBP1, GYG2, KAT2A, LGALS1, PFKP, and RGN ) were selected to develop a risk signature. There were significant differences in the clinical features (Fuhrman nuclear grade and TNM stage) between the high- and low-risk groups. The multivariable Cox regression indicated that the risk score was independent of the prognostic factors (training set: HR=3.393, 95% CI [2.025, 5.685], p<0.001; validation set: HR=1.933, 95% CI [1.130, 3.308], p=0.016). The AUCs of the nomograms for the 3-year OS in the training and validation sets were 0.808 and 0.819, respectively, and 0.777 and 0.796, respectively, for the 5- year OS. Conclusion: We demonstrated a novel glucose metabolism-related risk signature for predicting the prognosis of ccRCC. However, additional in vitro and in vivo research is required to validate our findings.

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