Enhancement of gemcitabine efficacy by K73-03 via epigenetically regulation of miR-421/SPINK1 in gemcitabine resistant pancreatic cancer cells.

BACKGROUND: Gemcitabine (GCB) is a first-line chemotherapeutic drug for pancreatic cancer (PCa). However, the resistance begins developing within weeks of chemotherapy. SPINK1 overexpression enhances resistance to chemotherapy. In a recent study, our laboratory established that the oleanolic acid (OA) derivative, K73-03, had a strong inhibitory effect on a SPINK1 overexpressed PCa cells. PURPOSE: In our current study, we studied the enhancement of GCB inhibitory effect by K73-03, a new novel OA derivative, alone or in combination with GCB on the GCB-resistant PCa cells by mitochondrial damage through regulation of the miR-421/SPINK1. METHODS: We detected the binding between miR-421 and SPINK1-3'-UTR in GCB-resistant PCa cells using Luciferase reporter assays. Cells viability, apoptosis, migration, and mitochondrial damage were investigated. RESULTS: The results demonstrated that the combination of K73-03 and GCB suppressed the growth of AsPC-1 and MIA PaCa-2 cells synergistically, with or without GCB resistance. Mechanistic findings showed that a combination of K73-03 and GCB silences SPINK1 epigenetically by miR-421 up-regulating, which leads to mitochondrial damage and inducing apoptosis in GCB-resistant PCa cells. CONCLUSION: We found an interesting finding that the 73-03 in combination with GCB can improve GCB efficacy and decrease PCa resistance, which induced apoptosis and mitochondrial damage through epigenetic inhibition of SPINK1 transcription by miR-421 up-regulation. This was the first study that used OA derivatives on GCB-resistant PCa cells, so this combined strategy warrants further investigation.

TL1A/TNFR2 Axis Enhances Immunoregulatory Effects of Bone Marrow Derived Mesenchymal Stem Cell by Indian Hedgehog Signaling Pathway.

BACKGROUND AND OBJECTIVES: The immunomodulatory potential of mesenchymal stem cells (MSCs) can be regulated by a variety of molecules, especially cytokines. The inflammatory cytokine, TNF-like ligand 1A (TL1A), has been reported as an inflammation stimulator in-multiple autoimmune diseases. Here, we studied the effects of TL1A/TNF-receptor 2 (TNFR2) pathway on the therapeutic potency of bone marrow-derived MSCs (BMSCs). METHODS AND RESULTS: BMSCs, fibroblast-like synoviocytes (FLSs), and H9 and jurkat human T lymphocytes were used in this study. BMSCs paracrine activities, differentiation, proliferation, and migration were investigated after stimulation with TL1A, and intervened with anti-TNFR2. Additionally, the effects of TL1A on BMSCs therapeutic potency were evaluated by treating RA-FLSs, and H9 and jurkat T cells with TL1A-stimulated BMSCs conditioned medium (CM). Indian hedgehog (IHH) involvement was determined by gene silencing and treatment by recombinant IHH (rIHH). TL1A induced BMSCs stemness-related genes, COX-2, IL-6, IDO, TGF-ß and HGF through TNFR2. Also, TL1A corrected biased differentiation and increased proliferation, and migration through TNFR2. Meanwhile, CM of TL1A-stimulated BMSCs decreased the inflammatory markers of RA-FLSs and T cells. Moreover, TL1A-stimulated BMSCs experienced IHH up-regulation coupled with NF-κB and STAT3 signaling up-regulation, while p53 and oxidative stress were down-regulated. Furthermore, treatment of BMSCs by rIHH increased their anti-inflammatory effects. More importantly, knockdown of IHH decreased the ability of TL1A-stimulated BMSCs to alleviating the inflammation in RA-FLSs and T cells. CONCLUSIONS: This study reports the effects of TL1A/TNFR2 pathway on the biological behaviors and therapeutic potency of BMSCs through IHH. These findings could introduce novel procedures to increase the stemness of MSCs in cellular therapy.

miR-421 up-regulation by the oleanolic acid derivative K73-03 regulates epigenetically SPINK1 transcription in pancreatic cancer cells leading to metabolic changes and enhanced apoptosis.

SPINK1 overexpression promotes cancer cell aggressiveness and confers chemo-resistance to multiple drugs in pancreatic cancer. Oleanolic acid (OA) derivatives possess active effects against different cancers. Here we report the effect of K73-03, a new novel OA derivative, against pancreatic cancer through mitochondrial dysfunction via miR-421/SPINK1 regulation. We examined the binding ability of miR-421 with SPINK1-3'UTR Luciferase reporter assays. Moreover, miR-421/SPINK1 expressions in pancreatic cancer, with or without K73-03 treatment, were evaluated. Cells viability, migration, autophagy, mitochondrial function and apoptosis were examined with or without K73-03 treatment. We established that the K73-03 effect on the miR-421 that plays a crucial role in the regulation of SPINK1 in pancreatic cancer. Our findings indicated that K73-03 inhibited the mitochondrial function that led to inducing autophagy and apoptosis through epigenetic SPINK1 down-regulation via miR-421 up-regulation in pancreatic cancer. Furthermore, the inhibition of miR-421 expression in pancreatic cancer cells abolished the efficacy of K73-03 against SPINK1 oncogenic properties. We found an interesting finding that the interaction between miR-421 and SPINK1 is related to mitochondrial function through the effect of K73-03. Further, SPINK1 appear to be the molecular targets of K73-03 especially more than gemcitabine.

Ezrin expression is altered in mice lymphatic metastatic hepatocellular carcinoma and subcellular fractions upon Annexin 7 modulation in-vitro.

Ezrin and Annexin seven (A7) have been suggested to be involved in several roles in cancers metastasis. However, the role of Ezrin and the effect of A7 on Ezrin expression in lymphatic metastatic hepatocellular carcinoma (LNM-HCC) have not been extensively explored yet. This study reports expression of Ezrin in high lymphatic metastasis (Hca-F >70%) and low metastatic metastasis (Hca-P <30%) HCC cell lines, and the effect of A7 on Ezrin expression. Real-Time PCR, Western blot, Subcellular fractionation, Immunocytochemistry and Immunofluorescence were used to investigate Ezrin expression in addition to migration and invasion behaviors of A7 up-regulated Hca-F cells, A7 down-regulated Hca-P and in their respective negative control (NC) cells. Ezrin expression was higher in high LNM-HCC than low LNM-HCC (p=0.0046). Cell fractionation analysis reveals that Ezrin was highly present in the cytoplasm, nucleus and cytoskeleton of NC-Hca-F cells. However, Ezrin was highly observed in the cell membrane, nucleus and cytoskeleton of NC-Hca-P cells. A7 up-regulation in Hca-F suppressed Ezrin expression (p=0.0248), but increase the migration and invasion, whereas Ezrin was mainly located in the cytoplasm and nucleus fractions. Down-regulation of A7 in Hca-P cells, enhanced Ezrin expression (p<0.0001) in the cytoplasm and nucleus fractions, and suppressed migration and invasion. In conclusion, Ezrin may play a role in LNM-HCC and might be inversely associated with A7 expression. The subcellular localization of Ezrin and A7 was varied according to the metastatic levels. Ezrin may thus be a potential diagnostic and/or prognostic biomarker for HCC.