Curcumin increases crizotinib sensitivity through the inactivation of autophagy via epigenetic modulation of the miR-142-5p/Ulk1 axis in non-small cell lung cancer.

Drug resistance is a critical factor responsible for the recurrence of non-small cell lung cancer (NSCLC). Previous studies suggest that curcumin acts as a chemosensitizer and radiosensitizer in human malignancies, but the underlying mechanism remains elusive. In the present study, we explored how curcumin regulates the expression of miR-142-5p and sensitizes NSCLC cells to crizotinib. We found that miR-142-5p is significantly downregulated in NSCLC tissue samples and cell lines. Curcumin could increase crizotinib cytotoxicity by epigenetically restoring the expression of miR-142-5p. Furthermore, curcumin treatment suppressed the expression of DNA methylation-related enzymes, including DNMT1, DNMT3A, and DNMT3B, in NSCLC cells. In addition, the upregulation of miR-142-5p expression increased crizotinib cytotoxicity and induced apoptosis in tumor cells in a similar manner to that of curcumin. Strikingly, miR-142-5p overexpression suppressed crizotinib-induced autophagy in A549 and H460 cells. Mechanistically, miR-142-5p inhibited autophagy in lung cancer cells by targeting Ulk1. Overexpression of Ulk1 abrogated the miR-142-5p-induced elevation of crizotinib cytotoxicity in A549 and H460 cells. Collectively, our findings demonstrate that curcumin sensitizes NSCLC cells to crizotinib by inactivating autophagy through the regulation of miR-142-5p and its target Ulk1.

Methylation­associated inactivation of LATS1 and its effect on demethylation or overexpression on YAP and cell biological function in human renal cell carcinoma.

Large tumor suppressor 1 (LATS1) gene is one of the key factors in Hippo signaling pathway. Inactivation of LATS1 by promoter methylation was found in colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), astrocytoma, breast cancer and it was proved to be a tumor suppressor. However, its role is unclear in renal cell carcinoma (RCC). In this study, the expression of LATS1 was determined by reverse transcription polymerase chain reaction (RT­PCR) and immunohistochemistry in 30 pairs of RCC tissues and matched normal kidney tissues and RCC cells. We found that the expression of LATS1 was markedly reduced in RCC tissues and cells, in the RCC tissue in 46.7% (14/30), while in the normal kidney tissues in 76.7% (23/30), and was associated with pathological grade and clinical stage of RCC. We detected methylation status of LATS1 by bisulfite sequence-PCR (BSP) in renal cancer cell line 786-O which lowers expression of LATS1, and we found it hypermethy-lated (in 97.5%). In addition, pharmacological demethylation using 5-Aza-2'-deoxycytidine (5-Aza) restored the expression of LATS1 mRNA and protein in 786-O cells, both LATS1 demethylation and overexpression of LATS1 downregulated the expression of Yes-associated protein (YAP), inhibited cell proliferation, induced cell apoptosis and cell cycle G1 arrest in 786-O cells. Thus, this report for the first time demonstrates the inactivation of LATS1 by promoter methy-lation and it is a tumor suppressor in kidney cancer. LATS1 may serve as a biomarker for possible early diagnosis and as a potential therapeutic target for human RCC.

YAP is overexpressed in clear cell renal cell carcinoma and its knockdown reduces cell proliferation and induces cell cycle arrest and apoptosis.

Yes-associated protein (YAP) has been reported to be an oncogene in a number of malignancies. It constitutes an important regulatory mechanism for the Hippo pathway, a key regulator of cell growth and apoptosis. The present study aimed to investigate the clinical significance and the role of YAP in the development of clear cell renal cell carcinoma (ccRCC). YAP expression levels were compared between ccRCC and adjacent normal renal tissues by RT-PCR and immunohistochemistry, respectively. YAP expression levels were then detected in ccRCC cell lines 786-0 and ACHN, as well as in human embryonic kidney 293 cells (HEK-293) using western blotting. Three specific YAP-shRNA lentiviral vectors were constructed and transfected into 786-0 cells, and then the mRNA and protein levels of YAP and downstream transcription factor TEAD1 were detected. Finally, the effects of YAP silencing on proliferation and the cell cycle distribution of 786-0 cells were detected by Cell Counting Kit-8 (CCK-8) and flow cytometry (FCM), respectively. The apoptosis rate was also analyzed by FCM. It was observed that the expression levels of YAP mRNA and protein in ccRCC tissues were higher than these levels in the adjacent normal renal tissues. The expression of YAP protein in ccRCC tissues was significantly correlated with clinical stage and differentiation. The YAP protein levels in the two ccRCC cell lines 786-0 and ACHN were significantly higher than that in the HEK-293 cells. Additionally, treatment of 786-0 cells with YAP-shRNA lentiviral vectors significantly reduced the expression levels of YAP and TEAD1 mRNA and protein. Further analyses in 786-0 cells in which YAP was decreased, revealed that cell proliferation was inhibited, cell cycle was arrested at the G1 phase and apoptosis was increased. These results indicate that YAP is an underlying oncogene in ccRCC and it may be a promising biomarker and therapeutic target of ccRCC.

Integrin-linked kinase functions as a tumor promoter in bladder transitional cell carcinoma.

The aim of this study was to elucidate the role of the integrin-linked kinase (ILK) gene in development of human bladder transitional cell carcinoma (BTCC). Expression of ILK protein and ILK mRNA in 56 cases of human BTCC tissue and in 30 cases of adjacent normal bladder tissue was detected by immunohistochemistry S-P and reverse transcription polymerase chain reaction (RT-PCR), respectively. Four specific miRNA RNAi vectors targeting human ILK were synthesized and transfected into BIU-87 cells by liposome to obtain stable expression cell strains. The influence of ILK on proliferation of BTCC was detected by MTT, FCM on athymic mouse tumorigenesis. The positive rate of ILK protein in BTCC tissue (53.6%) was much higher than adjacent normal bladder tissue (10.0%) (p<0.05). Similarly, expression of ILK mRNA in BTCC tissue (0.540 ± 0.083) was significantly higher than in adjacent normal bladder tissue (0.492 ± 0.070) (p<0.05). MTT showed that the proliferation ability of miRNA-ILK transfected group was clearly decreased (p<0.05), the cell cycle being arrested in G0/G1-S, an tumorigenesis in vivo was also significantly reduced (p<0.05). ILK gene transcription and protein expression may be involved in the development of BTCC, so that ILK might be the new marker for early diagnosis and the new target for gene treatment.

[Incidence rate and acupuncture-moxibustion treatment of post-stroke depression].

OBJECTIVE: To understand the incidence of post-stroke depression (PSD) and the therapeutic effect of acupuncture-moxibustion. METHODS: Five hundred and sixty cases were rating with Hamilton depression scale, and lesion parts, gender, age and property of stroke between the PSD and non-PSD were compared by analysis of variance, chi2 test, and then the patients of PSD were randomly divided into two groups and treated respectively with Prozac-20 and acup-moxibustion at Sishencong (EX-HN 1), Anmian, Neiguan (PC 6), Shenmen (HT 7), Zusanli (ST 36), Sanyinjiao (SP 6), Taichong (LR 3), Zhaohai (KI 6), Shenmai (BL 62). RESULTS: The total incidence rate of PSD was 43.9%, with no relation to the lesion parts, gender, age and property of stroke (P > 0.05), and there were very significant differences in HAMD scores before and after treatment in the two groups (P < 0.0001), with no significant difference in the effective rate between the two groups (P > 0.05). CONCLUSION: PSD has a high incidence and influences the recovery of nervous function, and it should be treated at early stage. Acup-moxibustion and Prozac-20 have similar therapeutic effect.