Xianjun Qu

Silencing of MicroRNA-21 confers the sensitivity to tamoxifen and fulvestrant by enhancing autophagic cell death through inhibition of the PI3K-AKT-mTOR pathway in breast cancer cells

Tamoxifen (TAM) and fulvestrant (FUL) represent the major adjuvant therapy to estrogen receptor-alpha positive (ER(+)) breast cancer patients. However, endocrine resistance to TAM and FUL is a great impediment for successful treatment. We hypothesized that miR-21 might alter the sensitivity of breast cancer cells to TAM or FUL by regulating cell autophagy. Using the ER(+) breast cancer cells, we knockdown miR-21.by transfection with miR-21 inhibitor, then the cells were exposed to TAM or FUL and the percentages of apoptosis and autophagy were determined. Knockdown of miR-21 significantly increased the TAM or FUL-induced apoptosis in ER(+) breast cancer cells. Further, silencing of miR-21 in MCF-7 cells enhanced cell autophagy at both basal and TAM or FUL-induced level. The increase of autophagy in miR-21-knockdown MCF-7 cells was also indicated by increase of beclin-1, LC3-II and increased GFP-LC3 dots. Importantly, knockdown of miR-21 contributed to autophagic cell death, which is responsible for part of TAM induced cell death in miR-21 inhibitor-transfected cells. Further analysis suggested that miR-21 inhibitor enhance autophagic cell death through inhibition of PI3K-AKT-mTOR pathway. MiR-21 coordinated the function of autophagy and apoptosis by targeting Phosphatase and tensin homolog (PTEN) through inhibition of PI3K-AKT-mTOR pathway. In conclusion, silencing of miR-21 increased the sensitivity of ER(+) breast cancer cells to TAM or FUL by increasing autophagic cell death. Targeting autophagy-related miRNAs is a potential strategy for overcoming endocrine resistance to TAM and FUL.

Resveratrol Inhibits the Invasion of Glioblastoma-Initiating Cells via Down-Regulation of the PI3K/Akt/NF-κB Signaling Pathway.

Invasion and metastasis of glioblastoma-initiating cells (GICs) are thought to be responsible for the progression and recurrence of glioblastoma multiforme (GBM). A safe drug that can be applied during the rest period of temozolomide (TMZ) maintenance cycles would greatly improve the prognosis of GBM patients by inhibiting GIC invasion. Resveratrol (RES) is a natural compound that exhibits anti-invasion properties in multiple tumor cell lines. The current study aimed to evaluate whether RES can inhibit GIC invasion in vitro and in vivo. GICs were identified using CD133 and Nestin immunofluorescence staining and tumorigenesis in non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. Invasive behaviors, including the adhesion, invasion and migration of GICs, were determined by tumor invasive assays in vitro and in vivo. The activity of matrix metalloproteinases (MMPs) was measured by the gelatin zymography assay. Western blotting analysis and immunofluorescence staining were used to determine the expression of signaling effectors in GICs. We demonstrated that RES suppressed the adhesion, invasion and migration of GICs in vitro and in vivo. Moreover, we proved that RES inhibited the invasion of GICs via the inhibition of PI3K/Akt/NF-κB signal transduction and the subsequent suppression of MMP-2 expression.

CXCL12/CXCR4 axis induced miR-125b promotes invasion and confers 5-fluorouracil resistance through enhancing autophagy in colorectal cancer

The activation of CXCL12/CXCR4 axis is associated with potential progression of cancer, such as invasion, metastasis and chemoresistance. However, the underlying mechanisms of CXCL12/CXCR4 axis and cancer progression have been poorly explored. We hypothesized that miRNAs might be critical downstream mediators of CXCL12/CXCR4 axis involved in cancer invasion and chemoresistance in CRC. In human CRC cells, we found that the activation of CXCL12/CXCR4 axis promoted epithelial-mesenchymal transition (EMT) and concurrent upregulation of miR-125b. Overexpression of miR-125b robustly triggered EMT and cancer invasion, which in turn enhanced the expression of CXCR4. Importantly, the reciprocal positive feedback loop between CXCR4 and miR-125b further activated the Wnt/β-catenin signaling by targeting Adenomatous polyposis coli (APC) gene. There was a negative correlation of the expression of miR-125b with APC mRNA in paired human colorectal tissue specimens. Further experiments indicated a role of miR-125b in conferring 5-fluorouracil (5-FU) resistance in CRC probably through increasing autophagy both in vitro and in vivo. MiR-125b functions as an important downstream mediator upon the activation of CXCL12/CXCR4 axis that involved in EMT, invasion and 5-FU resistance of CRC. These findings shed a new insight into the role of miR-125b and provide a potential therapeutic target in CRC.

Des-gamma-carboxy prothrombin antagonizes the effects of Sorafenib on human hepatocellular carcinoma through activation of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways

Despite significant progress, advanced hepatocellular carcinoma (HCC) remains an incurable disease, and the overall efficacy of targeted therapy by Sorafenib remains moderate. We hypothesized that DCP (des-gamma-carboxy prothrombin), a prothrombin precursor produced in HCC, might be one of the reasons linked to the low efficacy of Sorafenib. We evaluated the efficacy of Sorafenib in HLE and SK-Hep cells, both of which are known DCP-negative HCC cell lines. In the absence of DCP, Sorafenib effectively inhibited the growth of HCC and induced cancer cell apoptosis. In the presence of DCP, HCC was resistant to Sorafenib-induced inhibition and apoptosis, as determined by in vitro assays and in mice xenografted with HLE cells. Molecular analysis of HLE xenografted-nude mice showed that DCP activates the transduction of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR cascades. DCP might stimulate the formation of compensatory feedback loops in the intricately connected signaling pathways when kinases are targeted by Sorafenib. Our results indicate that DCP antagonizes the inhibitory effects of Sorafenib on HCC through activation of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR signaling pathways. Taken together, our findings define a DCP-mediated mechanism of inhibition of Sorafenib in HCC, which is critical for targeting therapy in advanced HCC.

Resveratrol sensitizes glioblastoma-initiating cells to temozolomide by inducing cell apoptosis and promoting differentiation.

Glioblastoma-initiating cells play crucial roles in the origin, growth, and recurrence of glioblastoma multiforme. The elimination of glioblastoma-initiating cells is believed to be a key strategy for achieving long-term survival of glioblastoma patients due to the highly resistant property of glioblastoma-initiating cells to temozolomide. Resveratrol, a naturally occurring polyphenol, has been widely studied as a promising candidate for cancer prevention and treatment. Whether resveratrol could enhance the sensitivity of glioblastoma-initiating cells to temozolomide therapy has not yet been reported. Here, using patient-derived glioblastoma-initiating cell lines, we found that resveratrol sensitized glioblastoma-initiating cells to temozolomide both in vitro and in vivo. Furthermore, we showed that resveratrol enhanced glioblastoma-initiating cells to temozolomide-induced apoptosis through DNA double-stranded breaks/pATM/pATR/p53 pathway activation, and promoted glioblastoma-initiating cell differentiation involving p-STAT3 inactivation. Our results propose that temozolomide and resveratrol combination strategy may be effective in the management of glioblastoma patients, particularly for those patients who have been present with a high abundance of glioblastoma-initiating cells in their tumors and show slight responsiveness to temozolomide.

Overexpression of SphK2 contributes to ATRA resistance in colon cancer through rapid degradation of cytoplasmic RXRα by K48/K63-linked polyubiquitination

The resistance mechanisms that limit the efficacy of retinoid therapy in cancer are poorly understood. Sphingosine kinase 2 (SphK2) is a highly conserved enzyme that is mainly located in the nucleus and endoplasmic reticulum. Unlike well-studied sphingosine kinase 1 (SphK1) located in the cytosol, little has yet understood the functions of SphK2. Here we show that SphK2 overexpression contributes to the resistance of all-trans retinoic acid (ATRA) therapy in colon cancer through rapid degradation of cytoplasmic retinoid X receptor α (RXRα) by lysine 48 (K48)- and lysine 63 (K63)-based polyubiquitination. Human colonic adenocarcinoma HCT-116 cells transfected with SphK2 (HCT-116Sphk2 cells) demonstrate resistance to ATRA therapy as determined by in vitro and in vivo assays. Sphk2 overexpression increases the ATRA-induced nuclear RXRα export to cytoplasm and then rapidly degrades RXRα through the polyubiquitination pathway. We further show that Sphk2 activates the ubiquitin-proteasome system through the signal mechanisms of (1) K48-linked proteosomal degradation and (2) K63-linked ubiquitin-dependent autophagic degradation. These results provide new insights into the biological functions of Sphk2 and the molecular mechanisms that underlie the Sphk2-mediated resistance to retinoid therapy.

Chemoprevention of intestinal tumorigenesis by the natural dietary flavonoid myricetin in APC Min/+ mice

Myricetin is a natural dietary flavonoid compound. We evaluated the efficacy of myricetin against intestinal tumorigenesis in adenomatous polyposis coli multiple intestinal neoplasia (APCMin/+) mice. Myricetin was given orally once a day for 12 consecutive weeks. APCMin/+ mice fed with myricetin developed fewer and smaller polyps without any adverse effects. Histopathological analysis showed a decreased number of dysplastic cells and degree of dysplasia in each polyp. Immunohistochemical and western blot analysis revealed that myricetin selectively inhibits cell proliferation and induces apoptosis in adenomatous polyps. The effects of myricetin were associated with a modulation the GSK-3β and Wnt/β-catenin pathways. ELISA analysis showed a reduced concentration of pro-inflammatory cytokines IL-6 and PGE2 in blood, which were elevated in APCMin/+ mice. The effect of myricetin treatment was more prominent in the adenomatous polyps originating in the colon. Further studies showed that myricetin downregulates the phosphorylated p38 MAPK/Akt/mTOR signaling pathways, which may be the mechanisms for the inhibition of adenomatous polyps by myricetin. Taken together, our data show that myricetin inhibits intestinal tumorigenesis through a collection of biological activities. Given these results, we suggest that myricetin could be used preventatively to reduce the risk of developing colon cancers.

Knockdown of CXCR4 Inhibits CXCL12-Induced Angiogenesis in HUVECs through Downregulation of the MAPK/ERK and PI3K/AKT and the Wnt/β-Catenin Pathways

ABSTRACT CXCL12 is an extracellular chemokine binding to cell surface receptor CXCR4. We found that activation of CXCL12/CXCR4 axis stimulated angiogenesis in endothelial cells. Knockdown of CXCR4 in endothelial cells prevented the branch points of angiogenesis. Endothelial cells exposed to CXCL12 presented high level of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and matrix metalloproteinase MMP-2, but not in CXCR4 knockdown cells. Further studies revealed that activation of CXCL12/CXCR4 axis in vascular endothelial cells stimulates the angiogenesis through upregulation of the MAPK/ERK and PI3K/AKT and Wnt/β-catenin pathways. Conclusion, downregulation of CXCR4 could inhibit angiogenesis in cancer tissues.

Chemopreventive effect of Myricetin, a natural occurring compound, on colonic chronic inflammation and inflammation-driven tumorigenesis in mice

Myricetin is a flavonoids compound extracted from edible myrica rubra. We aimed to evaluate the efficacy of Myricetin on colonic chronic inflammation and inflammation-driven tumorigenesis in mice. Myricetin was administrated by gavage for 4 consecutive weeks. Mice were sacrificed and the number of colonic polyps was counted. Myricetin significantly inhibited AOM/DSS-induced colitis and colorectal tumorigenesis. Myricetin prevented the incidence of colorectal tumorigenesis and reduced the size of colorectal polyps. Histopathologic analysis showed that Myricetin could attenuate the degree of colonic inflammation and colorectal tumorigenesis. Further analysis showed that Myricetin strongly reduced the levels of inflammatory factors TNF-α, IL-1β, IL-6, NF-κB, p-NF-κB, cyclooxygenase-2 (COX-2), PCNA and Cyclin D1 in the colonic tissues as analyzed by the assays of immunohistochemical staining, Western blotting and Q-RT-PCR. Our results demonstrated that Myricetin possesses the biological activities of chemoprevention colonic chronic inflammation and inflammation-driven tumorigenesis. We suggest that Myricetin could be developed as a promising chemopreventive drug for reducing the risk of colorectal cancer.

{2-[1-(3-Methoxycarbonylmethyl-1H-indol-2-yl)-1-methyl-ethyl]-1H-indol-3-yl}-acetic acid methyl ester (MIAM) inhibited human hepatocellular carcinoma growth through upregulation of Sirtuin-3 (SIRT3).

{2-[1-(3-Methoxycarbonylmethyl-1H-indol-2-yl)-1-methyl-ethyl]-1H-indol-3-yl}-acetic acid methyl ester (MIAM) is a novel indole compound. Our previous studies showed that MIAM possessed activity against many cancers xenografted in mice without significant toxicity. In this study, we determined the effect of MIAM on human hepatocellular carcinoma (HCC) by both in vitro and in vivo assays. In in vitro assay, the experiments were performed in the hypoxic incubator. MIAM inhibited HCC growth with dose-dependent manner. The effects of MIAM on HCC might be due to its activities in induction of apoptosis, arrest of cell cycle in G0/G1 phase. Further studies showed that MIAM might exert its actions through multiple mechanisms. MIAM could reduce intracellular ATP, increase levels of p53/p21 and SIRT3/SOD2/Bax. MIAM also had the activity of reducing HIF1α and hexokinase II (HK II) in HCC. MIAM had the activity of increasing cellular reactive oxygen species (ROS) in HCC. However, the increase of ROS might not be its main mechanism in inhibition of HCC. MIAM might inhibit HepG2 growth through induction of apoptosis. We determined the relationship between level of SIRT3 and cell viability in the MIAM-treated cells. MIAM treatment resulted in increase of SIRT3 in HCC. Further, HepG2 cells infected with human SIRT3 were more sensitive to MIAM than the cells without infection of SIRT3. These results suggested that MIAM might inhibit HCC growth through upregulation of SIRT3. Importantly, the effect of MIAM was confirmed in the HepG2 xenografts bearing in mice. MIAM treatment did not induce significant toxicology to mice. Together, MIAM could be developed as potential agent for treatment of HCC.