Mu-Xin Wei

Activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in cultured human colon cancer cells.

Here we report that activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in both primary cultured human colon cancer cells and cell lines. Knocking-down of AMPKα by the target shRNA significantly inhibits plumbagin-induced cytotoxicity in cultured colon cancer cells, while forced activation of AMPK by introducing a constitutively active AMPK (CA-AMPK), or by the AMPK activator, inhibits HT-29 colon cancer cell growth. Our Western-blots and immunoprecipitation (IP) results demonstrate that plumbagin induces AMPK/Apoptosis signal regulating kinase 1 (ASK1)/TNF receptor-associated factor 2 (TRAF2) association to activate pro-apoptotic c-Jun N-terminal kinases (JNK)-p53 signal axis. Further, after plumbagin treatment, activated AMPK directly phosphorylates Raptor to inhibit mTOR complex 1 (mTORC1) activation and Bcl-2 expression in colon cancer cells. Finally, we found that exogenously-added short-chain ceramide (C6) enhances plumbagin-induced AMPK activation and facilitates cell apoptosis and growth inhibition. Our results suggest that AMPK might be the key mediator of plumbagins anti-tumor activity.

MicroRNA-451 regulates AMPK/mTORC1 signaling and fascin1 expression in HT-29 colorectal cancer.

The earlier studies have shown that Fascin1 (FSCN1), the actin bundling protein, is over-expressed in colorectal cancers, and is associated with cancer cell progression. Here, we aimed to understand the molecular mechanisms regulating FSCN1 expression by focusing on mammalian target of rapamycin (mTOR) signaling and its regulator microRNA-451. We found that microRNA-451 was over-expressed in multiple colorectal cancer tissues, and its expression was correlated with mTOR complex 1 (mTORC1) activity and FSCN1 expression. In cultured colorectal cancer HT-29 cells, knockdown of FSCN1 by RNAi inhibited cell migration and proliferation. Activation of mTORC1 was required for FSCN1 expression, HT-29 cell migration and proliferation, as RAD001 and rapamycin, two mTORC1 inhibitors, suppressed FSCN1 expression, HT-29 cell migration and proliferation. Meanwhile, forced activation of AMP-activated protein kinase (AMPK), the negative regulator of mTORC1, by its activators or by the genetic mutation, inhibited mTORC1 activation, FSCN1 expression, cell migration and proliferation. In HT-29 cells, we found that over-expression of microRNA-451 inhibited AMPK activation, causing mTORC1 over-activation and FSCN1 up-regulation, cells were with high migration ability and proliferation rate. Significantly, these effects by microRNA-451 were largely inhibited by mTORC1 inhibitors or the AMPK activator AICAR. On the other hand, knockdown of miRNA-451 by the treatment of HT-29 cells with miRNA-451 antagomir inhibited mTORC1 activation and FSCN1 expression. The proliferation and migration of HT-29 cells after miRNA-45 knockdown were also inhibited. Our results suggested that the over-expressed microRNA-451 in colon cancer cells might inhibit AMPK to activate mTORC1, which mediates FSCN1 expression and cancer cell progression.

Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

Here we evaluated the anti-cancer activity of aqueous Oldenlandia diffusa (OD) extracts (ODE) in colorectal cancer (CRC) cells. We showed that ODE exerted potent anti-proliferative, cytotoxic and pro-apoptotic activities against a panel of established CRC lines (HCT-116, DLD-1, HT-29 and Lovo) and primary (patient-derived) human CRC cells. ODE activated AMP-activated protein kinase (AMPK) signaling, which led to subsequent mTORC1 inhibition and Bcl-2/HIF-1α downregulation in CRC cells. In ODE-treated CRC cells, AMPKα1 formed a complex with p53. This might be important for p53 activation and subsequent cancer cell apoptosis. Inhibition of AMPK signaling, though dominant negative (dn) mutation or shRNA/siRNA knockdown of AMPKα1 attenuated ODE-exerted CRC cytotoxicity. In vivo, i.p. administration of ODE inhibited HCT-116 xenograft tumor growth in SCID mice. In addition, AMPK activation, mTORC1 inhibition and p53 activation were observed in ODE-treated HCT-116 xenograft tumors. These results suggest that ODE inhibits CRC cells in vitro and in vivo, possibly via activation of AMPK-dependent signalings.

C6 ceramide dramatically increases vincristine sensitivity both in vivo and in vitro, involving AMP-activated protein kinase–p53 signaling

Use of the conventional cancer chemotherapy (i.e. vincristine) is limited in tumor cells exhibiting pre-existing or acquired resistance. Here, we found that C6 ceramide (C6) dramatically sensitized vincristines activity. In vitro, C6 and vincristine coadministration induced substantial necrosis and apoptosis in multiple human cancer cell lines, which were accompanied by a profound AMP-activated protein kinase (AMPK) activation, subsequent p53 activation, mTORC1 inactivation and Bcl-2/HIF-1α downregulation. Such synergistic effects were attenuated by AMPK inactivation through genetic mutation or short hairpin RNA silencing. Coadministration-activated p53 translocated to mitochondria, and formed a complex with cyclophilin-D, leading to mitochondrial permeability transition pore opening and cell necrosis. Disrupting p53-Cyp-D complexation through pharmacological or genetic means reduced costimulation-induced cytotoxicity. In vivo, a liposomal C6 was synthesized, which dramatically enhanced the antiproliferative activity of vincristine on HCT-116 or A2780 xenografts. Together, C6 sensitizes vincristine-induced anticancer activity in vivo and in vitro, involving activating AMPK-p53 signaling.

Association between polymorphisms of trinucleotide repeat containing 9 gene and breast cancer risk: evidence from 62,005 subjects.

Trinucleotide repeat containing 9 (TNRC9) is a gene located at chromosome 16q12. Although of an uncertain function, it is a newly described risk factor for breast cancer. It contains a putative high-mobility group box motif, suggesting its possible role as transcription factor; it has been implicated in breast cancer metastasis. Published studies on the association between TNRC9 polymorphisms and breast cancer risk remain inconclusive, and a meta-analysis is required to verify the association. This pioneering research performed a meta-analysis of eight studies comprising a total of 25,828 cases and 36,177 controls. Significantly elevated breast cancer risk was associated with TNRC9 rs3803662 polymorphism when all studies were pooled in the meta-analysis (T vs. C allele contrast model: OR 1.18, 95% CI 1.09–1.28; TT vs. CC homozygote codominant model: OR 1.26, 95% CI 1.02–1.55; TT vs. CC+CT recessive model: OR 1.23, 95% CI 1.06–1.42). For TNRC9 rs12443621 polymorphism, no significant association was detected in all genetic models. For TNRC9 rs12443621 polymorphism, meanwhile, no significant association was observed in all comparison models. Conclusively, this meta-analysis suggests that TNRC9 rs3803662 polymorphism was significantly correlated with breast cancer risk and the variant T allele of TNRC9 rs3803662 polymorphism is a low-penetrant risk factor for developing breast cancer. There is no significant association between TNRC9 rs12443621 and rs8051542 polymorphisms and risk of breast cancer in current literature.

KU-0060648 inhibits hepatocellular carcinoma cells through DNA-PKcs-dependent and DNA-PKcs-independent mechanisms

Here we tested anti-tumor activity of KU-0060648 in preclinical hepatocellular carcinoma (HCC) models. Our results demonstrated that KU-0060648 was anti-proliferative and pro-apoptotic in established (HepG2, Huh-7 and KYN-2 lines) and primary human HCC cells, but was non-cytotoxic to non-cancerous HL-7702 hepatocytes. DNA-PKcs (DNA-activated protein kinase catalytic subunit) is an important but not exclusive target of KU-0060648. DNA-PKcs knockdown or dominant negative mutation inhibited HCC cell proliferation. On the other hand, overexpression of wild-type DNA-PKcs enhanced HepG2 cell proliferation. Importantly, KU-0060648 was still cytotoxic to DNA-PKcs-silenced or -mutated HepG2 cells, although its activity in these cells was relatively weak. Further studies showed that KU-0060648 inhibited PI3K-AKT-mTOR activation, independent of DNA-PKcs. Introduction of constitutively-active AKT1 (CA-AKT1) restored AKT-mTOR activation after KU-0060648 treatment in HepG2 cells, and alleviated subsequent cytotoxicity. In vivo, intraperitoneal (i.p.) injection of KU-0060648 significantly inhibited HepG2 xenograft growth in nude mice. AKT-mTOR activation was also inhibited in xenografted tumors. Finally, we showed that DNA-PKcs expression was significantly upregulated in human HCC tissues. Yet miRNA-101, an anti-DNA-PKcs miRNA, was downregulated. Over-expression of miR-101 in HepG2 cells inhibited DNA-PKcs expression and cell proliferation. Together, these results indicate that KU-0060648 inhibits HCC cells through DNA-PKcs-dependent and -independent mechanisms.

Association between mitogen-activated protein kinase kinase kinase 1 rs889312 polymorphism and breast cancer risk: evidence from 59,977 subjects

Published data on the association between mitogen-activated protein kinase kinase kinase 1 (MAP3K1) gene rs889312 polymorphism and breast cancer risk are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. Crude ORs with 95% CIs were used to assess the strength of association between them. A total of seven eligible articles including 26,015 cases and 33,962 controls based on the search criteria were involved in this meta-analysis. We observed that the MAP3K1 rs889312 polymorphism was significantly correlated with breast cancer risk from the fixed effects model when all studies were pooled into the meta-analysis (the allele contrast model: OR 1.09, 95% CI 1.07–1.12; the homozygote codominant: OR 1.22, 95% CI 1.15–1.29; the heterozygote codominant: OR 1.07, 95% CI 1.04–1.11; the dominant model: OR 1.10, 95% CI 1.06–1.13; the recessive model: OR 1.18, 95% CI 1.12–1.25). No significant association was found in the BRCA1 mutation carriers in all genetic models. When stratified by BRCA2 mutation carriers status, statistically significantly elevated risk was found in this meta-analysis (C vs. A: OR 1.12, 95% CI 1.01–1.23; CC vs. AA: OR 1.35, 95% CI 1.06–1.71; the recessive model: OR 1.31, 95% CI 1.05–1.65). There was no evidence for significant association between MAP3K1 rs889312 polymorphism and breast cancer risk in BRCA1 and BRCA2 positive cohort for all comparison models. In conclusion, this meta-analysis suggests that the MAP3K1 rs889312 C allele is a low-penetrant risk factor for developing breast cancer, and there is limited evidence to indicate that MAP3K1 rs889312 polymorphism is associated with increased risk of breast cancer in BRCA1 mutation carriers.

Identification of sphingosine kinase 1 (SphK1) as a primary target of icaritin in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a highly aggressive neoplasm. We aim to explore the anti-HCC activity by a natural prenylflavonoid icaritin. Icaritin was cytotoxic and pro-apoptotic when added to established (HepG2, KYN-2 and Huh-7 lines) and primary human HCC cells. At the signaling level, icaritin inhibited sphingosine kinase 1 (SphK1) activity in HCC cells, which led to pro-apoptotic ceramide production and JNK1 activation. SphK1 inhibition or silence (by shRNA/microRNA) mimicked icaritin-mediated cytotoxicity, and almost nullified icaritins activity in HepG2 cells. Reversely, exogenous over-expression of SphK1 sensitized icaritin-induced HepG2 cell apoptosis. In vivo, oral administration of icaritin dramatically inhibited HepG2 xenograft growth in SCID mice. Further, SphK1 activity in icaritin-treated tumors was largely inhibited. In summary, icaritin exerts potent anti-HCC activity in vitro and in vivo. SphK1 inhibition could be the primary mechanism of its actions in HCC cells.

Need for clarification of data in a recent meta-analysis about TP53 codon 72 polymorphism and cancer susceptibility

Pei-Hua Lu ,y, Mu-Xin Wei2,y, Chen Li3,y, Wei Shen and Min-Bin Chen Department of General Surgery, Wuxi People’s Hospital of Nanjing Medical University, No. 299, Qingyang Road, Wuxi 214023, Jiangsu, China, Department of Traditional Chinese Medicine, First Affiliated Hospital of Nanjing Medical University, No.300, Guangzhou Road, Nanjing 210029, Jiangsu, China, Department of Gastroenterology, Xuzhou Chinese Medicine Hospital Affiliated to Nanjing University of Chinese Medicine, No.80, Zhongshan South Road, Xuzhou 221000, Jiangsu, China and Department of Oncology, Kunshan People’s Hospital Affiliated to Jiangsu University, No. 91, Qianjin Road, Kunshan 215300, Jiangsu, China

No significant association results obtained from significant association evidence: the ongoing uncertainty of TP53 codon 72 polymorphism and breast cancer risk

1.063, 95% CI 0.967–1.169; Arg/Arg vs. Pro/Pro: OR 1.245, 95% CI 0.997–1.554; dominant model: OR 1.146, 95% CI 0.979–1.340; recessive model: OR 1.179, 95% CI 1.020–1.362). Actually, the odds ratio of 1.179 and 95% confidence interval for odds ratio of 1.020–1.362 in recessive model demonstrated some evidence of association between TP53 codon 72 polymorphism and breast cancer risk. Similarly, Ma et al. [1] demonstrated that no significant association was observed for any of the genetic models in the stratified analysis by source of controls (Pro/Arg vs. Pro/Pro: OR 1.075, 95% CI 0.974–1.187; Arg/Arg vs. Pro/ Pro: OR 1.283, 95% CI 1.036–1.588; dominant model: OR 1.158, 95% CI 1.007–1.333; recessive model: OR 1.229, 95% CI 1.054–1.433). Obviously, significant associations were observed in the Arg/Arg vs. Pro/Pro, the dominant and the recessive models. Thus, the ongoing uncertainty still existed and the conclusion by Ma et al. [1] was not entirely credible. Importantly, several sizeable eligible studies (7,601 cases and 11,549 controls) have not been included in this meta-analysis [2–16], even though they satisfied the search criteria. Of note, there were 5 studies of Asians [2–6], 8 studies of Caucasians [7–14], and 2 studies of mixed