Adamantyl Retinoid-Related Molecules Induce Apoptosis in Pancreatic Cancer Cells by Inhibiting IGF-1R and Wnt/β-Catenin Pathways

Pancreatic carcinoma has a dismal prognosis as it often presents as locally advanced or metastatic. We have found that exposure to adamantyl-substituted retinoid-related (ARR) compounds 3-Cl-AHPC and AHP3 resulted in growth inhibition and apoptosis induction in PANC-1, Capan-2, and MiaPaCa-2 pancreatic cancer cell lines. In addition, AHP3 and 3-Cl-AHPC inhibited growth and induced apoptosis in spheres derived from the CD44+/CD24+ (CD133+/EpCAM+) stem-like cell population isolated from the pancreatic cancer cell lines. 3-Cl-AHPC-induced apoptosis was preceded by decreasing expression of IGF-1R, cyclin D1, β-catenin, and activated Notch-1 in the pancreatic cancer cell lines. Decreased IGF-1R expression inhibited PANC-1 proliferation, enhanced 3-Cl-AHPC-mediated apoptosis, and significantly decreased sphere formation. 3-Cl-AHPC inhibited the Wnt/β-catenin pathway as indicated by decreased β-catenin nuclear localization and inhibited Wnt/β-catenin activation of transcription factor TCF/LEF. Knockdown of β-catenin using sh-RNA also induced apoptosis and inhibited growth in pancreatic cancer cells. Thus, 3-Cl-AHPC and AHP3 induce apoptosis in pancreatic cancer cells and cancer stem-like cells and may serve as an important potential therapeutic agent in the treatment of pancreatic cancer

A novel mechanism of lncRNA and miRNA interaction: CCAT2 regulates miR-145 expression by suppressing its maturation process in colon cancer cells

Abstract Background Although both long and micro RNAs are emerging as important functional components in colorectal cancer (CRC) progression and metastasis, the mechanism of their interaction remains poorly understood. CCAT2 (Colon cancer-associated transcript-2), a long noncoding RNA (lncRNA), has been reported to be over-expressed in CRC and is found to promote tumor growth. miRNAs, a class of naturally occurring short RNAs negatively control the expression of target genes by cleaving mRNA or through translation repression. Recently, we reported that miR-145 and miR-21 cooperate to regulate colon cancer stem cell (CSC) proliferation and differentiation. Considering that CCAT2 is mainly located in the nucleus and miRNA maturation process begins in the nucleus, we hypothesize that CCAT2 selectively blocks miR-145 maturation process, resulting in decreased mature miR-145 affecting colon CSC proliferation and differentiation. Methods The levels of CCAT2 were manipulated by transfection of CCAT2 expression plasmid or knockdown by siRNA or by CRISPR/Cas9. Quantitative RT-PCR was performed to examine the expression of CCAT2 and pri-, pre- and mature miR-145/21. Fluorescence in situ hybridization (FISH) was used to visualize CCAT2 in the cells. In vitro processing of pri-miRNA-145 was performed using T7 RNA polymerase and recombinant human Dicer. Results We have observed that modulated expression of CCAT2 regulates the expression of miR-145 in colon cancer HCT-116 and HT-29 cells. Knockout of CCAT2 increases miR-145 and negatively regulates miR-21 in HCT-116 cells, impairs proliferation and differentiation. In contrast, stable up-regulation of CCAT2 decreases mature miR-145 and increases the expression of several CSC markers in colon cancer cells. We have also observed that CCAT2 is enriched in the nucleus and correlates with the expression of pre-miR-145 but not pre-miR-21 in HCT-116 cells. These results indicate CCAT2 selectively blocks miR-145 maturation by inhibiting pre-miR-145 export to cytoplasm. Further, we revealed that CCAT2 blocks cleavage of pre-miR-145 by Dicer in vitro. Conclusions Our results identify CCAT2 as a negative regulator of miRNA-145 biogenesis, and expose a novel mechanism of lncRNA-miRNA crosstalk

Additional file 1: Figure S1. of A novel mechanism of lncRNA and miRNA interaction: CCAT2 regulates miR-145 expression by suppressing its maturation process in colon cancer cells

Additional Figure and legend. (DOCX 560 kb

Upregulation of miR-150* and miR-630 induces apoptosis in pancreatic cancer cells by targeting IGF-1R.

MicroRNAs have been implicated in many critical cellular processes including apoptosis. We have previously found that apoptosis in pancreatic cancer cells was induced by adamantyl retinoid-related (ARR) molecule 3-Cl-AHPC. Here we report that 3-Cl-AHPC-dependent apoptosis involves regulating a number of microRNAs including miR-150* and miR-630. 3-Cl-AHPC stimulated miR-150* expression and caused decreased expression of c-Myb and IGF-1R in the pancreatic cancer cells. 3-Cl-AHPC-mediated reduction of c-Myb resulted in diminished binding of c-Myb with IGF-1R and Bcl-2 promoters, thereby causing repression of their transcription and protein expression. Over-expression of miR-150* also resulted in diminished levels of c-Myb and Bcl-2 proteins. Furthermore, the addition of the miRNA inhibitor 2'-O-methylated miR-150 blocked 3-Cl-AHPC-mediated increase in miR-150* levels and abrogated loss of c-Myb protein. Knockdown of c-Myb in PANC-1 cells resulted in enhanced apoptosis both in the presence or absence of 3-Cl-AHPC confirming the anti-apoptotic property of c-Myb. Overexpression of miR-630 also induced apoptosis in the pancreatic cancer cells and inhibited target protein IGF-1R mRNA and protein expression. Together these results implicate key roles for miR-150* and miR-630 and their targeting of IGF-1R to promote apoptosis in pancreatic cancer cells

Metformin: a potential therapeutic agent for recurrent colon cancer.

Accumulating evidence suggests that metformin, a biguanide class of anti-diabetic drugs, possesses anti-cancer properties. However, most of the studies to evaluate therapeutic efficacy of metformin have been on primary cancer. No information is available whether metformin could be effectively used for recurrent cancer, specifically colorectal cancer (CRC) that affects up to 50% of patients treated by conventional chemotherapies. Although the reasons for recurrence are not fully understood, it is thought to be due to re-emergence of chemotherapy-resistant cancer stem/stem-like cells (CSCs/CSLCs). Therefore, development of non-toxic treatment strategies targeting CSCs would be of significant therapeutic benefit. In the current investigation, we have examined the effectiveness of metformin, in combination with 5-fluorouracil and oxaliplatin (FuOx), the mainstay of colon cancer therapeutics, on survival of chemo-resistant colon cancer cells that are highly enriched in CSCs/CSLCs. Our data show that metformin acts synergistically with FuOx to (a) induce cell death in chemo resistant (CR) HT-29 and HCT-116 colon cancer cells, (b) inhibit colonospheres formation and (c) enhance colonospheres disintegration. In vitro cell culture studies have further demonstrated that the combinatorial treatment inhibits migration of CR colon cancer cells. These changes were associated with increased miRNA 145 and reduction in miRNA 21. Wnt/β-catenin signaling pathway was also down-regulated indicating its pivotal role in regulating the growth of CR colon cancer cells. Data from SCID mice xenograft model of CR HCT-116 and CR HT-29 cells show that the combination of metformin and FuOX is highly effective in inhibiting the growth of colon tumors as evidenced by ∼ 50% inhibition in growth following 5 weeks of combination treatment, when compared with the vehicle treated controls. Our current data suggest that metformin together with conventional chemotherapy could be an effective treatment regimen for recurring colorectal cancer (CRC)

Natural agents inhibit colon cancer cell proliferation and alter microbial diversity in mice.

The current study was undertaken to investigate the effect of differentially formulated polyphenolic compound Essential Turmeric Oil-Curcumin (ETO-Cur), and Tocotrienol-rich fraction (TRF) of vitamin E isomers on colorectal cancer (CRC) cells that produce aggressive tumors. Combinations of ETO-Cur and TRF were used to determine the combinatorial effects of ETO-Cur and TRF-mediated inhibition of growth of CRC cells in vitro and HCT-116 cells xenograft in SCID mice. 16S rRNA gene sequence profiling was performed to determine the outcome of gut microbial communities in mice feces between control and ETO-Cur-TRF groups. Bacterial identifications were validated by performing SYBR-based Real Time (RT) PCR. For metagenomics analysis to characterize the microbial communities, multiple software/tools were used, including Quantitative Insights into Microbial Ecology (QIIME) processing tool. We found ETO-Cur and TRF to synergize and that the combination of ETO-Cur-TRF significantly inhibited growth of HCT-116 xenografts in SCID mice. This was associated with a marked alteration in microbial communities and increased microbial OTU (operation taxonomic unit) number. The relative abundance of taxa was increased and the level of microbial diversity after 34 days of combinatorial treatment was found to be 44% higher over the control. Shifting of microbial family composition was observed in ETO-Cur-TRF treated mice as evidenced by marked reductions in Bacteroidaceae, Ruminococcaceae, Clostridiales, Firmicutes and Parabacteroids families, compared to controls. Interestingly, during the inhibition of tumor growth in ETO-Cur treated mice, probiotic Lactobacillaceae and Bifidobacteriaceae were increased by 20-fold and 6-fold, respectively. The relative abundance of anti-inflammatory Clostridium XIVa was also increased in ETO-Cur-TRF treated mice when compared with the control. Our data suggest that ETO-Cur-TRF show synergistic effects in inhibiting colorectal cancer cell proliferation in vitro and in mouse xenografts in vivo, and might induce changes in microbial diversity in mice

Inhibitor of miR-150* blocked 3-Cl-AHPC-mediated decreased of c-Myb in PANC-1 cells.

<p>(A) Inhibitor 2′-O-methylated miR-150* (OME-150) inhibited 3-Cl-AHPC mediated miR-150* expression in transiently transfected cells. (B and C) miR-150 inhibitor blocked the 3-Cl-AHPC mediated c-Myb degradation. Cells were treated with 1 µM 3-Cl-AHPC for 24 h. (D) Knock down of c-Myb expression in cells. (E) Knock down of c-Myb enhanced the apoptosis in cells. Cells stably transfected with three sh-Myb knockdown expression vectors and scrumble sequence control vector were exposed to 3-Cl-AHPC for 24 h. Induction of apoptosis was assessed using Annexin V-FITC labeling with propidium iodide (PI) staining in cells and the error bars represent the mean of three separate determinations ± the standard deviation (SD). All treated samples and sh-Myb are significantly different from control and sh-vector; • and ••, ** (<0.05 and <0.01 respectively) are significantly different by <i>t</i>-Test and • represented the comparison between 3-Cl-AHPC to OME-150*+3-Cl-AHPC treated samples.</p