Pazopanib stimulates senescence of renal carcinoma cells through targeting nuclear factor E2-related factor 2 (Nrf2).

Renal cell carcinoma (RCC) is the most common kidney cancer with high mortality rate. Pazopanib has been approved for the treatment of RCC. However, the underlying mechanism is not clear. Here, we report a novel finding by showing that treatment with Pazopanib could promote cellular senescence of the human RCC cell line ACHN. Cells were stimulated with 5, 10, and 20 µM Pazopanib, respectively. Cellular senescence was measured using senescence-associated ß-galactosidase (SA-ß-Gal) staining. Western blot analysis and real-time polymerase chain reaction were used to measure the mRNA and protein expression of nuclear factor E2-related factor 2 (Nrf2), γH2AX, human telomerase reverse transcriptase (hTERT), telomeric repeat binding factor 2 (TERF2), p53 and plasminogen activator inhibitor (PAI). First, we found that exposure to Pazopanib reduced the cell viability of ACHN cells. Additionally, Pazopanib induced oxidative stress  by increasing the production of reactive oxygen species, reducing the levels of glutathione peroxidase, and promoting nuclear translocation of Nrf2. Interestingly, Pazopanib exposure resulted in DNA damage by increasing the expression of γH2AX. Importantly, Pazopanib increased cellular senescence and reduced telomerase activity. Pazopanib also reduced the gene expression of hTERT but increased the gene expression of TERF2. Correspondingly, we found that Pazopanib increased the expression of p53 and PAI at both the mRNA and protein levels. To elucidate the underlying mechanism, the expression of Nrf2 was knocked down by transduction with Ad- Nrf2 shRNA. Results indicate that silencing of Nrf2 in ACHN cells abolished the effects of Pazopanib in stimulating cellular senescence and reducing telomerase activity. Consistently, knockdown of Nrf2 restored the expression of p53 and PAI in ACHN cells. Based on these results, we explored a novel mechanism whereby which Pazopanib displays a cytotoxicity effect in RCC cells through promoting cellular senescence mediated by Nrf2.

Initial experience with surrounding en bloc transurethral resection of bladder tumor and simultaneous intravesical treating for non-muscle invasive bladder cancer.

PURPOSE: The high recurrence rate after traditional transurethral resection of bladder tumor (TURBT) remains a challenge for management of non-muscle invasive bladder tumor (NMIBC). The aim of this study was to evaluate feasibility, efficacy and safety of surrounding en bloc resection using a general wire bipolar loop electrode and simultaneous intravesical chemotherapy. METHODS: We retrospectively analyzed data of 111 consecutive patients with NMIBC treated from June 2018 to December 2021. These patients underwent conventional TURBT and immediate intravesical chemotherapy (n = 45) or surrounding en bloc TURBT and simultaneous intravesical chemotherapy in the Urology Department of Harbin Medical University Cancer Hospital, The former and latter were defined as the conventional TURBT group and the surrounding en bloc TURBT group, respectively. All patients were followed up from 6 to 40 months, with an average of 24 months. Demographic characteristics, location and number of tumors, perioperative and postoperative data, pathological results and recurrence were documented. RESULTS: There were no significant differences in clinicopathological data between the conventional TURBT group (n = 45) and the surrounding en bloc TURBT group (n = 66). Operative time and complications associated with TURBT were comparable in the two groups. Recurrent tumors were found during follow-up in 2 (3.0%) of 66 patients in the surrounding en bloc group and 9 (20%) of 45 patients in the conventional group (p < 0.05). Lower urinary tract symptoms developed in 2 (3.0%) of 66 patients after surrounding en bloc TURBT and in 11(24.4%) of 45 patients after conventional TURBT (p < 0.05). CONCLUSION: Surrounding en bloc TURBT and simultaneous intravesical chemotherapy might significantly decrease the recurrence rate of NMIBC, and showed favorable safety and tolerability profiles. The general bipolar loop electrode was appropriate to complete the procedure.

MiRNA-148a inhibits cell growth and drug resistance by regulating WNT10a expression in renal cell carcinoma.

Background: We aimed to explore miR-148a exerts a tumor suppressor effect and arsenic trioxide (As2O3) sensitivity on renal cell carcinoma (RCC). Methods: We performed polymerase chain reaction (PCR) on 42 pairs of tumor and paracancerous samples collected from RCC patients to investigate the miR-148a expression; meanwhile, we analyzed the interplay between clinical indicators and miR-148a expression of RCC. Then, the influence of miR-148a overexpression on the functions of RCC cells were analyze using transwell migration assay, Cell Counting Kit-8 (CCK-8), and cell wound healing assay. Furthermore, the ability of miR-148a to sensitize Caki-1 cells treated with As2O3 were detected using flow cytometry. Finally, the relevant mechanism of miR-148a on the downstream gene Wnt family member 10A (WNT10a) was explored by cell reverse method. Results: The results from RCC patients indicated a significantly lower miR-148a level than adjacent tissues. The low miR-148a expression increased prevalence of distant metastasis and decreased survival rate compared to those with high expression in patients. In the RCC cell lines, the proliferation and metastasis ability of the miR-148a mimic group was remarkably lower than the miR-NC group. At the same time, it was verified that WNT10a was remarkably higher cell lines and RCC tissues; and negatively related to miR-148a expression. In addition, miR-148a mimics were found to remarkably reduce the protein expression of WNT10a. In the cell reverse experiment, overexpression of WNT10a was confirmed to offset the miR-148a mimics effect on metastasis and proliferation of RCC cells. In addition, an increase in relative apoptosis was detected in As2O3 treated with/without miR-148a mimics for 48 hours, and apoptosis was significantly reduced after transfection with WNT10a in the Caki-1 cell line and significantly reduced after combined treatment. Conclusions: The study revealed that miR-148a is associated with distant metastases and leads to poor prognosis in RCC patients. Moreover, miR-148a inhibit the malignant progression and increase the sensitivity of RCC cells to As2O3 by regulating WNT10a.

Long Noncoding RNA KCNMB2-AS1 Promotes SMAD5 by Targeting miR-3194-3p to Induce Bladder Cancer Progression.

PURPOSE: Bladder cancer is a common malignant tumor of the urinary system, with the fourth-highest incidence of male malignant tumors in Europe and the United States. So far, the mechanism of bladder cancer progression and metastasis has not been clarified. The aim of our study was to validate the way of long noncoding RNA (lncRNA) KCNMB2-AS1 on the metabolism and growth of bladder cancer cells by miR-3194-3p/SMAD5. PATIENTS AND METHODS: The Gene Expression was analyzed by qRT-PCR in bladder cancer tissues and cell lines, with the highly expressed KCNMB2-AS1 screened out. Cell proliferation was detected by Edu staining and clone formation assay, cell migration, and invasion by wound healing and transwell assays. Cell stemness was determined by assessing sphere-forming ability and stemness marker. Correlation between miRNA and lncRNA/gene was verified by dual-luciferase assay and RIP, and the effect of KCNMB2-AS1 on bladder cancer growth by nude mice tumor formation experiment. RESULTS: Here, we revealed the increased level of KCNMB2-AS1 in bladder cancer for the first time. Knockdown of KCNMB2-AS1 in vitro prevented the ability of proliferation, metastasis, and stemness of cancer cells. In vivo, the silencing of KCNMB2-AS1 also prevented tumor growth in vivo. Next, we revealed that KCNMB2-AS1 could interact with miR-3194-3p and uncovered that SAMD5 was a downstream target of miR-3194-3p. CONCLUSION: In conclusion, KCNMB2-AS1 mediated the bladder cancer cells progress by regulating the miR-3194-3p/SAMD5 signal pathway, which would provide a new target for bladder cancer research.

LncRNA HOTAIR induces sunitinib resistance in renal cancer by acting as a competing endogenous RNA to regulate autophagy of renal cells.

BACKGROUND: Cell autophagy has been proposed to be involved in drug resistance therapy. However, how the long non-coding RNA (lncRNA) reduces risks of drug resistance in renal cancer (RC) cells needs a thorough inquiry. This study was assigned to probe the effect and mechanism of HOTAIR on sunitinib resistance of RC. METHODS: Clinical RC tissues and para-carcinoma tissues were obtained to detect the expressions of miR-17-5p, HOTAIR and Beclin1. Sunitinib-resistant cells (786-O-R and ACHN-R) were constructed using parental RC cells (786-O and ACHN). The resistance of 786-O-R and ACHN-R cells to sunitinib was examined. Western blot and qRT-PCR were assayed to obtain the expressions of miR-17-5p, HOTAIR and Beclin1. The effects of HOTAIR knockdown or miR-17-5p overexpression/knockdown on cell autophagy and sunitinib resistance were measured by MDC staining, immunofluorescence and Western blot. The sensitivity of RC cells to sunitinib and change in cell clone formation after sunitinib treatment were assessed by CCK-8 assay and colony formation assay, respectively. The relationships among HOTAIR, miR-17-5p and Beclin1 were verified by dual-luciferase reporter gene and RIP assay. The role of HOTAIR knockdown in sunitinib resistance was verified in nude mice. RESULTS: HOTAIR expression in sunitinib-resistant cells is higher than that in parental cells. Knockdown of HOTAIR in sunitinib-resistant cells lead to refrained sunitinib resistance and cell autophagy both in vivo and in vitro. Activation of autophagy could raise resistance to sunitinib in RC cells, while inhibition of autophagy could improve the sensitivity of sunitinib-resistant cells to sunitinib. HOTAIR could compete with miR-17-5p to regulate Beclin1 expression. Knockdown of miR-17-5p in parental cells increases cell resistant to sunitinib, and overexpression of miR-17-5p in sunitinib-resistant cells increases cell sensitive to sunitinib. CONCLUSION: HOTAIR negatively targets miR-17-5p to activate Beclin1-mediated cell autophagy, thereby enhancing sunitinib resistance in RC cells.

Disruption of por1 gene in Candida utilis improves co-production of S-adenosylmethionine and glutathione.

The por1 gene encoding one of the mitochondrial porin channels in C. utilis CCTCC M 209298 was disrupted using a homologous recombination method. The co-production of S-adenosylmethionine (SAM) and glutathione (GSH) in the mutant C. utilis Δpor1 increased by 34.9% and 25.1%, respectively, during batch and fed-batch fermentation, relative to the parental strain. The average oxygen consumption rate, activities of key enzymes involved in SAM and GSH biosynthesis, levels of intracellular cofactors such as NADH and ATP, and carbon fluxes of key metabolites were compared between the parental strain and the Δpor1 mutant. The disruption of por1 gene increased the rate of mitochondrial respiration, increased the activities of both methionine adenosyltransferase and γ-glutamylcysteine synthetase, and enhanced the supply of energy and substrates for SAM and GSH biosynthesis, all of which favored the overproduction of SAM and GSH in the Δpor1 mutant.

Caspase-10, matrix metalloproteinase-9 and total laminin are correlated with the tumor malignancy of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a common malignant kidney tumor, the pathogenesis of which remains unclear. The aim of the present study was to investigate whether caspase-10, matrix metalloproteinase-9 (MMP-9) and total laminin (LM) were involved into the pathogenesis of ccRCC. The levels of caspase-10, MMP-9 and total LM were analyzed by ELISA in tumor tissues and adjacent non-malignant tissues of 27 patients with ccRCC. The results revealed that caspase-10 levels in the tumor tissues were significantly higher than those in the adjacent non-malignant tissues (P<0.05). The MMP-9 levels in the tumor tissues were significantly lower than those in adjacent non-malignant tissues (P<0.01). The total LM levels in tumor tissues revealed no statistical difference with those in the adjacent non-malignant tissues (P=0.757). Additionally, caspase-10 levels were positively correlated with MMP-9 levels (P<0.001), but negatively correlated with total LM levels (P<0.05) in tumor tissues. Correlation analyses with clinical data of patients with ccRCC, revealed that caspase-10 levels (P<0.05) and MMP-9 levels (P<0.001) in tumor tissues were positively correlated with tumor grades of ccRCC, whereas total LM levels were positively correlated with tumor size (P<0.05). The results of the present study suggested that interactions between caspase-10, MMP-9 and LM are likely involved in the pathogenesis of ccRCC. A deeper understanding of the correlation between caspase-10, MMP-9 and LM would aid the clarification of pathogenesis of ccRCC.

Design, synthesis and biological activities of tetrandrine and fangchinoline derivatives as antitumer agents.

The isolation and modification of natural products is always a very important resources to anti-tumor drugs. Therefore, a novel series of tetrandrine and fangchinoline derivatives were designed and synthesized, and their antiproliferative activities against HepG2, MCF-7 cells were evaluated and described. From the activity result obtained, high to very high activity in vitro has been found, one of the tested compounds (compound 5d) exhibited the most significant cytotoxic effects. Compound 5d increased 29.2, 7.37 times anti-proliferative activity for HepG2 cells and MCF-7 cells compared to sunitinib (IC50=16.06µM and 25.41µM). Finally flow cytometry determined that compound 5d could indeed inhibit the proliferation of HepG2 cells via inducing apoptosis.

[Bone morphogenetic protein signal transduction pathway regulates runx2 expression in MC3T3 -E1 osteoblasts in vitro induced by centrifugation].

OBJECTIVE: To observe the expression of Runx2 in osteoblasts in response to centrifugation in vitro and discuss the function of bone morphogenetic protein (BMP) signal transduction pathway in this course. METHODS: Cells were divided into four groups, group A, B, C and D, pretreated with DMEM containing 10% fetal bovine serum, 10% fetal bovine serum, 100 ng x mL(-1) Noggin and 100 ng x mL(-1) Noggin for 24 hours separately. 271 x g centrifugation was loaded for 5 min to these groups except group A and C, other conditions were the same. The total RNA of each group were extracted, and reversed transcription to cDNA after 30 min. The expression of Runx2 in response to centrifugation in vitro was analyzed by quantitative real time PCR. RESULTS: The expression of Runx2 mRNA in group B was significantly higher than that in group A (P < 0.05). The expression of Runx2 mRNA in group D was significantly lower than that in group B (P < 0.05). There was no statistically significant difference among group A, C, D (P = 0.692). CONCLUSION: BMP signal transduction pathway plays an important role in the response of osteoblasts to mechanical stimulations. It may also play a central role in the cascade information dissemination of osteoblasts.

[Experimental study on the effect of hypoxia on the expression of VEGF and TGF-beta1 of rat mandibular osteoblasts].

OBJECTIVE: To observe the effects of cobalt chloride (CoCl2)-simulated hypoxia on VEGF and TGF-beta1 expression and to provide theoretical basis for deciphering the molecular mechanism of clinical distraction osteogenesis. METHODS: The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining, the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoCl2, and then immunofluorescence was used to detect the expressions of VEGF and TGF-beta1 at 0, 3, 6, 9, 12 and 24 hours after culture. RESULTS: The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing line was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outline was unclear, and at the cell compact district, massive masculine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that masculine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after 15 days of culture. After alizarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the laser confocal microscope. As the hypoxia time prolonged, VEGF fluorescence of cells in the experimental group intensified, and reached the peak 9 hours after operation, and then dropped to the normal level. At various time points, TGF-beta1 fluorescence was found in both groups under the laser confocal microscope, and fluorescence intensity in the control group was slightly stronger than that in the VEGF control group. In the experimental group, TGF-beta1 expression had short-term increase 3 hours after hypoxia, and reduced gradually with the prolonging of hypoxia time. CONCLUSION: The method of culturing osteoblast from Wistar rats mandibular is practicable. The cells can be used for further studies. Moderate hypoxia can affect bone synthesis and turnover in distraction osteogenesis and up-regulate the expressions of VEGF and TGF-beta1.

The involvement of hypoxia-inducible factor-1alpha in the susceptibility to gamma-rays and chemotherapeutic drugs of oral squamous cell carcinoma cells.

The transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) is the key regulator that controls the hypoxic response of mammalian cells. The overexpression of HIF-1alpha has been demonstrated in many human tumors. However, the role of HIF-1alpha in the therapeutic efficacy of chemotherapy and radiotherapy in cancer cells is poorly understood. In this study, we investigated the influence of HIF-1alpha expression on the susceptibility of oral squamous cell carcinoma (OSCC) cells to chemotherapeutic drugs (cis-diamminedichloroplatinum and 5-fluorouracil) and gamma-rays. Treatment with chemotherapeutic drugs and gamma-rays enhanced the expression and nuclear translocation of HIF-1alpha, and the susceptibility of OSCC cells to the drugs and gamma-rays was negatively correlated with the expression level of HIF-1alpha protein. The overexpression of HIF-1alpha induced OSCC cells to become more resistant to the anticancer agents, and down-regulation of HIF-1alpha expression by small interfering RNA enhanced the susceptibility of OSCC cells to them. In the HIF-1alpha-knockdown OSCC cells, the expression of P-glycoprotein, heme oxygenase-1, manganese-superoxide dismutase and ceruloplasmin were downregulated and the intracellular levels of chemotherapeutic drugs and reactive oxygen species were sustained at higher levels after the treatment with the anticancer agents. These results suggest that enhanced HIF-1alpha expression is related to the resistance of tumor cells to chemo- and radio-therapy and that HIF-1alpha is an effective therapeutic target for cancer treatment.

Mechanism of HIF-1alpha-dependent suppression of hypoxia-induced apoptosis in squamous cell carcinoma cells.

The transcriptional factor hypoxia-inducible factor-1 (HIF-1) plays an important role in solid tumor cell growth and survival. Overexpression of HIF-1alpha has been demonstrated in many human tumors and predicts a poor response to chemoradiotherapy. We examined the HIF-1alpha-induced survival pathways in human oral squamous cell carcinoma cell (OSCC) lines. The results showed that forced expression of HIF-1alpha suppressed hypoxia-induced apoptosis of OSCC lines by inhibiting cytochrome c release from mitochondria. Overexpression of HIF-1alpha inhibited the generation of reactive oxygen species (ROS), elevation of intracellular Ca(2+) concentration, reduction of mitochondrial membrane potential, and cytosolic accumulation of cytochrome c, which resulted in the inactivation of caspase-9 and caspase-3. In addition, antiapoptotic Bcl-2 and Bcl-X(L) levels were increased and pro-apoptotic Bax and Bak levels were decreased in the HIF-1alpha-overexpressing OSCC line. Overexpression of HIF-1alpha also increased the levels of phosphorylation of Akt and extracellular signal-regulated kinases (ERK). These findings indicate that HIF-1alpha prevents apoptotic cell death through two mechanisms, including inhibition of cytochrome c release and activation of Akt and ERK.

Reactive oxygen species (ROS) control the expression of Bcl-2 family proteins by regulating their phosphorylation and ubiquitination.

We examined the influence of ROS on the phosphorylation and complex formation of Bcl-2 family proteins in Mn-superoxide dismutase (SOD) antisense-transfected squamous cell carcinoma cells, OSC-4 cells. The increase of intracellular ROS level induced by cis-diamminedichloroplatinum (CDDP) and gamma-ray treatment was greater in antisense-transfected cells than in control vector-transfected cells, and apoptosis was more extensively induced in the former. Antisense-transfected cells expressed high levels of Bax and Bak, but low levels of Bcl-2 and Bcl-XL when treated with CDDP, peplomycin, 5-fluorouracil or gamma-rays. After treatment with these agents, the phosphorylation of protein kinase A, Bcl-2 (Thr56) and Bad (Ser155) was increased, especially in antioxidant (N-acetylcysteine and pyrrolidine dithiocarbamate)-pretreated control cells, but the phosphorylation levels were very low in the antisense-transfected cells. Bcl-2 ubiquitination was increased, but ubiquitination of Bad and Bax was decreased in the antisense-transfected cells, although their ubiquitination was increased by the antioxidants. These results reveal that ROS induce apoptosis by regulating the phosphorylation and ubiquitination of Bcl-2 family proteins, resulting in increased proapoptotic protein levels and decreased antiapoptotic protein expression.