Gallbladder Cancer Cell Proliferation Research Articles

Abstract 1916: Targeting adhesion signaling network blocks tumor growth in gallbladder cancer

Abstract 96 Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} Gallbladder cancer (GBC) is a highly fatal malignancy with a 5-year survival rate of 17.6%. The prognosis of GBC is poor due to the aggressive tumor biology, late presentation, complicated anatomic position, and advanced stage at diagnosis. Characterized by a poor response to chemotherapy and radiotherapy, locally advanced and metastatic disease in patients with GBC is often treated with chemotherapy with palliative purpose. Therefore, development of new effective therapies is urgently needed for the treatment of GBC. The aim of the current study is to evaluate whether triptolide and its water soluble analog Minnelide is effective against GBC cells. We have previously shown that triptolide/Minnelide reduces tumor growth in various cancer models via regulating epithelial -mesenchymal transition (EMT), an important mechanism underlying metastasis. In our preliminary findings using two GBC cell lines, GB-d1 and NOZ, we demonstrate that triptolide inhibits the proliferation of GBC cells. Mice harboring subcutaneous gallbladder tumors, treated with Minnelide, exhibited a significant reduction in tumor weights and volume. In order to elucidate the mechanism underlying triptolide/Minnelide mediated inhibition of cellular proliferation in GBC cells and reduction in tumor growth, we identified Aurora Kinase A (AURKA) and FAK as two new targets for triptolide action in GBC cells. Triptolide regulates the activity of AURKA and FAK via an adhesion signaling protein NEDD9 which regulates the activation of several oncogenic proteins including FAK and SRC. By targeting both FAK and AURKA, triptolide disrupts the integrity of focal adhesion structures via deregulating adhesion signaling network. In addition, by targeting AURKA in GBC cells triptolide inhibits the cap-dependent translation machinery via eukaryotic translation initiation factor 4E (eIF4E). Our preliminary findings regarding potential use of Minnelide in GBC are promising. <!–EndFragment–> Citation Format: Mahendra K. Singh, Kaustav Majumder, Bhuwan Giri, Nivedita Arora, Shrey Modi, Vrishketan Sethi, Claudia P. Garcia, Juan C. Roa, Vikas Dudeja, Sulagna Banerjee, Ashok K. Saluja. Targeting adhesion signaling network blocks tumor growth in gallbladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1916.

RETRACTED: EMP3, which is regulated by miR-663a, suppresses gallbladder cancer progression via interference with the MAPK/ERK pathway

Gallbladder cancer (GBC) is the most common malignancy of the biliary tract and its molecular pathogenesis is poorly understood. Aberrant expression of epithelial membrane protein-3 (EMP3) was reported in different kinds of cancers. Our study aimed to explore the elusive functional roles and the underlying molecular mechanisms of EMP3 with respect to GBC progression. The results showed that human GBC tissues exhibited decreased levels of EMP3 compared with non-malignant tissues. Kaplan-Meier analysis indicated that low expression of EMP3 was associated with poor prognosis of GBC patients. Upregulation of EMP3 repressed GBC cell proliferation, migration and invasion both in vitro and in vivo. Conversely, EMP3 silencing promoted GBC cell growth and metastasis. Additionally, we found that EMP3 was a target gene of miR-663a, and downregulation of EMP3 in GBC was attributed to the overexpression of miR-663a. Furthermore, miR-663a was proven to be a tumor-promoting factor mediating GBC development. Finally, we demonstrated that downregulation of EMP3 activated MAPK/ERK signaling, which modulated GBC progression. These data showed the mechanism by which EMP3 suppresses GBC progression, suggesting that the miR-663a/EMP3/MAPK/ERK axis may be a novel therapeutic target for GBC treatment.

Effect of CD147 inhibitor tunicamycin and activator angiotensin II on gallbladder carcinoma cells

Objective To research the effect of CD147 inhibitor tunicamycin and activator angiotensin Ⅱ (AngⅡ) on proliferation, invasion, and expression of CD147 and matrix metalloproteinase (MMP)-9 in GBC-SD cells. Methods GBC-SD cells were treated with different concentrations of tunicamycin (5, 10, 20 μl/ml), AngⅡ (5, 10, 20 μl/ml) alone or tunicamycin plus AngⅡ (tunicamycin 10 μl/ml + AngⅡ 20 μl/ml). The rate of proliferation was detected by cell counting kit-8 (CCK-8). The expression of CD147 and MMP-9 was detected by Western blotting. The ability of invasion was examined by Transwell assay. Results (1) As compared with the control group, proliferation rate of GBC-SD in tunicamycin group was significantly decreased in a time- and concentration-dependent manner [(88.17±8.36)% vs. (100.00±0.00)%, P=0.005; (85.35±6.42)% vs. (100.00±0.00)%, P=0.003; (70.15±6.89)% vs. (100.00±0.00)%, P=0.000]. As compared with the control group, the CD147 value of K was significantly decreased (0.75±0.04 vs. 0.88±0.07, P=0.046; 0.71±0.04 vs. 0.88±0.07, P=0.009), and MMP-9 value of K was also decreased (0.84±0.04 vs. 1.10±0.08, P=0.004; 0.67±0.10 vs. 1.10±0.08, P=0.000) in a concentration-dependent manner in in tunicamycin group. The invasive ability in tunicamycin group was significantly decreased as compared with control group (43.00±10.98 vs. 67.65±8.65, P=0.034; 32.46±7.71 vs. 67.65±8.65, P=0.005). (2) The cell proliferation rate in AngⅡ group was gradually increased in a time- and concentration-dependent manner [(104.32±5.05)% vs. (100.00±0.00)%, P=0.056; (108.25±6.57)% vs. (100.00±0.00)%, P=0.019; (115.50±8.65)% vs. (100.00±0.00)%, P=0.003], the CD147 value of K increased in a concentration-dependent manner (1.02±0.08 vs. 0.88±0.07, P=0.039; 1.08±0.05 vs. 0.88±0.07, P=0.005), MMP-9 value of K increased (1.28±0.06 vs. 1.10±0.08, P=0.034; 1.57±0.07 vs. 1.10±0.08, P=0.001), and the invasion ability increased (98.00±12.56 vs. 67.65±8.65, P=0.013) as compared with the control group. There was significant difference in cell invasion, CD147 expression and MMP-9 expression between the combined group and the control group. Conclusion Tunicamycin can inhibit the proliferation of gallbladder cancer cells, reduce the expression of CD147 and MMP-9, and AngⅡcan accelerate the proliferation of gallbladder cancer cells, and increase the expression of CD147 and MMP-9. The combined use of tunicamycin and AngⅡ can counteract the interaction. Key words: Gallbladder carcinoma cell; Tunicamycin; Angiotensin Ⅱ; CD147; Matrix metalloproteinase-9

Knockdown of TRIM31 suppresses proliferation and invasion of gallbladder cancer cells by down-regulating MMP2/9 through the PI3K/Akt signaling pathway

Tripartite motif (TRIM) 31, a member of the TRIM protein family, contributes to a wide range of biological processes and its altered expression exerts a non-negligible effect on multiple pathological conditions such as immunological disorders and retroviral protective activity. Recently, increasing evidence has demonstrated an important role of TRIM31 in the development of various cancers. However, the role of TRIM31 in gallbladder cancer (GBC) remains unclear. In this study, we showed that TRIM31 was elevated in GBC tissues and cell lines and associated with the clinicopathological features of GBC patients. Down-regulation of TRIM31 suppressed GBC cell proliferation, migration and invasion in vitro as well as inhibited tumor growth and metastasis in vivo. In addition, knockdown of TRIM31 reduced the expression of MMP2, MMP9 and p-Akt. Taken together, these findings indicated that knockdown of TRIM31 suppressed proliferation and invasion of GBC cells and was associated with PI3K/Akt signaling inactivation. Thus, TRIM31 may be a potential therapeutic target for the treatment of GBC.

LDHA is a direct target of miR-30d-5p and contributes to aggressive progression of gallbladder carcinoma.

Gallbladder cancer (GBC) is the most general biliary tract malignancy, with poor prognosis due to rapid tumor progression and lack of specific symptoms. Lactate dehydrogenase-A (LDHA) can promote Warburg effect to produce lactate and Adenosine Triphosphate (ATP) in aerobic condition, which contributes to oncogenesis metastasis and drug resistance in various cancers. However, the expression and functional role of LDHA in GBC are largely unknown. We determined that LDHA was over-expressed in GBC tumor tissues compared with normal tissues, which was also an independent prognostic factor for the overall survival of GBC patients by tissue microarrays analysis. In addition, RNAi-mediated LDHA silencing could suppress the GBC cell proliferation, invasion, colony formation and glycolysis while promoting cell apoptosis in vitro. Similar results were observed in GBC cells treated with LDHA specific inhibitor FX11. Moreover, we confirmed that knockdown of LDHA could inhibit tumor growth in vivo. Additionally, we found that the 3'-untranslated region (3'-UTR) of LDHA mRNA was the direct target of microRNA-30d-5p (miR-30d-5p), which was low expressed in GBC tissues and associated with poor prognosis of GBC patients. Our findings disclose a novel role for miR-30d-5p/LDHA axis contribute to aggressive progression by reprogramming the metabolic process in GBC cells, and suggest a potential application of miR-30d-5p/LDHA axis in prognosis prediction and GBC treatment.

MicroRNA-30a-5p inhibits gallbladder cancer cell proliferation, migration and metastasis by targeting E2F7

Gallbladder carcinoma (GBC), the most common malignant tumour of the bile duct, is highly aggressive and has a poor prognosis. MicroRNA-30a-5p (miR-30a-5p) is an important tumour suppressor that participates in many aspects of carcinogenesis and cancer development. However, the role of miR-30a-5p in GBC development remains to be determined, as do the mechanisms underlying its effects in GBC. Using samples collected from 42 subjects with gallbladder carcinoma (GBC), we showed decreased miR-30a-5p expression in the primary lesions vs. non-tumour adjacent tissues (NATs). Decreased miR-30a-5p was associated with shorter disease-free survival (DFS) and overall survival (OS). Inhibiting miR-30a-5p expression in 2 representative GBC cell lines (GBC-SD and NOZ) increased cell proliferation, migration, invasiveness, as well as β-catenin nuclear translocation, vice versa. In nude mice, NOZ cells transfected with miR-30a-5p mimics grew slower (vs. miR-NC) upon subcutaneous inoculation, and had lower rate of hepatic metastasis upon spleen inoculation. Dual luciferase assay confirmed that E2F transcription factor 7 (E2F7) was a direct target of miR-30a-5p and antagonized the effects induced by miR-30a-5p downregulation in GBC cells. MiR-30a-5p attenuates the EMT and metastasis in GBC cells by targeting E2F7, suggesting miR-30a-5p is a tumour suppressor that may serve as a novel potential prognostic biomarker or molecular therapeutic target for GBC.

Prognostic significance of long non-coding RNA MALAT1 for predicting the recurrence and metastasis of gallbladder cancer and its effect on cell proliferation, migration, invasion, and apoptosis.

The objective of this study is to explore the role of MALAT1 as a molecular indicator in predicting the recurrence, metastasis, and prognosis of gallbladder cancer (GBC) and its effect on the proliferation, invasion, migration, and apoptosis of GBC cells in vitro. GBC tissues and adjacent normal tissues were collected from 102 patients. MALAT1 short hairpin RNA (shRNA) plasmids were first constructed to transfect the GBC-SD cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was applied to detect MALAT1 expression. CCK-8 assay, flow cytometry, and Transwell assay were applied to testify the cell proliferation, cell cycle, apoptosis, invasion, and migration. A receiver operating characteristic (ROC) curve was used to evaluate the values of MALAT1 in GBC recurrence, metastasis, and prognosis. COX regression analysis was applied to analyze the independent influencing factors of GBC patients' survival status. ROC curve results showed that the MALAT1 expression could be a predictor of the GBC recurrence, metastasis, and prognosis. According to the COX regression analysis, MALAT1 expression, tumor size, and TNM stage were independent influencing factors of GBC patients' survival condition. Compared with the GBC-SD cells transfected with empty plasmids, those transfected with MALAT1 shRNA plasmids showed higher apoptosis rates, weakened proliferation, migration, and invasion. In conclusion, our findings demonstrate that lncRNA MALAT1 can be considered as an indicator for evaluating the recurrence, metastasis, and prognosis of GBC patients. We also demonstrate how the overexpression of MALAT1 confers an oncogenic function in GBC.

Metformin synergistically enhances antitumor activity of cisplatin in gallbladder cancer via the PI3K/AKT/ERK pathway.

Metformin (Met) is a widely used antidiabetic drug and has demonstrated interesting anticancer effects in various cancer models, alone or in combination with chemotherapeutic drugs. The aim of the present study is to investigate the synergistic effect of Met with cisplatin (Cis) on the tumor growth inhibition of gallbladder cancer cells (GBC-SD and SGC-996) and explore the underlying mechanism. Cells were treated with Met and/or Cis and subjected to cell viability, colony formation, apoptosis, cell cycle, western blotting, xenograft tumorigenicity assay and immunohistochemistry. The results demonstrated that Met and Cis inhibited the proliferation of gallbladder cancer cells, and combination treatment with Met and Cis resulted in a combination index<1, indicating a synergistic effect. Co-treatment with Met and Cis caused G0/G1 phase arrest by upregulating P21, P27 and downregulating CyclinD1, and induced apoptosis through decreasing the expression of p-PI3K, p-AKT, and p-ERK. In addition, pretreatment with a specific AKT activator (IGF-1) significantly neutralized the pro-apoptotic activity of Met+Cis, suggesting the key role of AKT in this process. More importantly, in nude mice model, Met and Cis in combination displayed more efficient inhibition of tumor weight and volume in the SGC-996 xenograft mouse model than Met or Cis alone. Immunohistochemistry analysis suggests the combinations greatly suppressed tumor proliferation, which is consistent with our in vitro results. In conclusion, our findings indicate that the combination therapy with Met and Cis exerted synergistic antitumor effects in gallbladder cancer cells through PI3K/AKT/ERK pathway, and combination treatment with Met and Cis would be a promising therapeutic strategy for gallbladder cancer patients.

Shikonin induces apoptosis and G0/G1phase arrest of gallbladder cancer cells via the JNK signaling pathway.

Shikonin, a natural product isolated from the roots of Lithospermumerythrorhizon, is considered to have antitumor effects. Gallbladder cancer (GBC) is a prevalent biliary tract malignancy with few curative therapeutic stragegies and poor prognosis. In the present study, we detected the effects of shikonin on GBC cells as well as the underlying molecular mechanisms. The results demonstrated that GBC cell proliferation was inhibited by shikonin as determined by MTT and colony formation assays. Flow cytometry results demonstrated that shikonin treatment enhanced apoptosis and promoted G0/G1phase arrest in the GBC cells. Western blot assay showed that shikonin induced mitochondrial-dependent apoptosis via the JNK signaling pathway. Moreover, shikonin suppressed tumor growth in mice bearing GBC-derived xenografts in a dose‑dependent manner without side-effects. These results revealed that shikonin exhibits anticancer effects on GBC cells by inducing apoptosis and regulating the cell cycle. Taken together, shikonin may be a novel and safe chemotherapeutic agent for the treatment of GBC.

Effect of CD147 antagonist peptide and doxycycline on gallbladder carcinoma cells

Objective To research the effect of GBC-SD on proliferation, apoptosis, expression of CD147 and activity of matrix metalloproteinase (MMP)-9 in gallbladder carcinoma cells, with single and combination therapy of CD147 antagonist peptide and doxycycline. Methods Gallbladder carcinoma cells were treated with antagonist peptide (AP-9) (50, 100, 200, 400 μg/ml) or DOX (5, 10, 20, 40 μg/ml) alone and combination therapy (AP-9 200 μg/ml + DOX 20 μg/ml) for 12, 24 and 48 h, then the inhibition rate of proliferation was detected by cell counting kit-8 (CCK-8), expression of CD147 was detected by Western blotting, the activity of MMP-9 was detected by gelatin zymography, and the early apoptosis rate was detected by flow cytometry with Annexin V/propidium iodide (PI) staining. Results After treatment with several concentrations of AP-9, the apoptosis rate [(2.13±0.10)% and (2.92±0.19)%] was higher (P=0.002, P=0.001) than control group [(1.58±0.08)%]; the activity of MMP-9 [(320.08±33.90), (249.59±33.63), (259.63±34.08) and (172.31±35.20)] was reduced (P=0.013, 0.002, 0.003 and 0.001 respectively) as compared with that in control group; and cell proliferation was inhibited as compared with that in control group (P=0.001). After treatment with several concentrations of doxycycline, the apoptosis rate [(3.25±0.12)% and (3.63±0.24)%] was higher (P=0.001) than in the control group [(1.58±0.08)%]; the activity of MMP-9 [(203.52±18.25), (175.22±23.30), (147.86±22.83) and (109.99±29.22)] was reduced (P=0.001) as compared with control group [(446.08±42.42)]; cell proliferation was inhibited as compared with control group (P=0.001); the expression level of CD147 [(0.97±0.12), (0.80±0.10), (0.69±0.11) and (0.51±0.12)] was reduced (P=0.001) as compared with cotrol group [(2.04±0.15)]. As compared with treatment alone, cell proliferation [(38.78±0.39)%, (43.76±0.71)% and (52.00±0.91)%] was increased (P=0.001, P=0.003), the activity of MMP-9 [(110.19±24.74)] was reduced (P=0.001, P=0.024), and the apoptosis rate [(3.73±0.29)%] was elevated after combination therapy (P=0.001, P=0.048). Conclusion DOX can down-regulate the expression of CD147. AP-9 and DOX alone or in combination can inhibit the proliferation of gallbladder cancer cells, inhibit the activity of MMP-9, and promote apoptosis, and the inhibitory effect was enhanced after combination therapy. Key words: Gallbladder carcinoma; CD147; Extracellular matrix metalloproteinase-9; Antagonist peptide; Doxycycline

EIF3D promotes gallbladder cancer development by stabilizing GRK2 kinase and activating PI3K-AKT signaling pathway

Recent evidence suggests that dysregulated eIF3d expression may be critical in various genetic disorders as well as cancer. In this study, we observed that EIF3d levels increased in gallbladder cancer (GBC) samples compared with non-tumor tissue. High eIF3d levels were associated with advanced tumor stage and metastasis and were correlated with poor prognosis in 92 patients with GBC. Depletion of EIF3d in GBC cell lines inhibited cell proliferation, colony formation and metastasis and induced apoptosis and cell cycle arrest in vitro and in vivo. In contrast, ectopic expression of eIF3d had the opposite effects. Moreover, in this study, we revealed that a novel non-translational factor function of eIF3d mediated its protumoral effects. In details, eIF3d stabilizes GRK2 protein by blocking ubiquitin-mediated degradation, consequently activates PI3K/Akt signaling, and promotes GBC cell proliferation and migration. In conclusion, eIF3d promotes GBC progression mainly via eIF3d–GRK2–AKT axis and it may be used as a prognostic factor. The therapeutic targeting of eIF3d–GRK2 axis may be a potential treatment approach for GBC.

Long non-coding RNA UCA1 promotes gallbladder cancer progression by epigenetically repressing p21 and E-cadherin expression.

A growing number of studies indicated that long non-coding RNAs (lncRNAs) determine some cellular processes in cancer, such as proliferation, metastasis and differentiation. Urothelial carcinoma associated 1 (UCA1), an lncRNA, had been reported for its overexpression and oncogenic effect on various human cancers. In this study, we found that UCA1 was significantly overexpressed in gallbladder cancer (GBC) and positively correlated with tumor size, lymph node metastasis, TNM stage and short survival time. Moreover, UCA1 promoted GBC cell proliferation and metastasis in vitro and tumor growth in vivo. Mechanically, we identified that UCA1 promoted GBC progression through recruiting enhancer of zeste homolog 2 (EZH2) to the promoter of p21 and E-cadherin, and epigenetically suppressing their transcript.

Chloride intracellular channel 1 regulates the antineoplastic effects of metformin in gallbladder cancer cells.

Metformin is the most commonly used drug for type 2 diabetes and has potential benefit in treating and preventing cancer. Previous studies indicated that membrane proteins can affect the antineoplastic effects of metformin and may be crucial in the field of cancer research. However, the antineoplastic effects of metformin and its mechanism in gallbladder cancer (GBC) remain largely unknown. In this study, the effects of metformin on GBC cell proliferation and viability were evaluated using the Cell Counting Kit‐8 (CCK‐8) assay and an apoptosis assay. Western blotting was performed to investigate related signaling pathways. Of note, inhibition, knockdown and upregulation of the membrane protein Chloride intracellular channel 1 (CLIC1) can affect GBC resistance in the presence of metformin. Our data demonstrated that metformin apparently inhibits the proliferation and viability of GBC cells. Metformin promoted cell apoptosis and increased the number of early apoptotic cells. We found that metformin can exert growth‐suppressive effects on these cell lines via inhibition of p‐Akt activity and the Bcl‐2 family. Notably, either dysfunction or downregulation of CLIC1 can partially decrease the antineoplastic effects of metformin while upregulation of CLIC1 can increase drug sensitivity. Our findings provide experimental evidence for using metformin as an antitumor treatment for gallbladder carcinoma.

Combination of celecoxib and PD184161 exerts synergistic inhibitory effects on gallbladder cancer cell proliferation.

Cyclooxygenase-2 (COX-2) and extracellular signal-regulated kinase 1/2 (ERK1/2) may serve as potential targets in various types of cancer; however, the roles of these proteins in gallbladder carcinoma (GBC) have not been reported previously. In the present study, the expression levels of COX-2 and phospho (p)-ERK1/2 in GBC were examined and the biological activities of celecoxib and PD184161 (specific inhibitors of COX-2 and p-ERK1/2, respectively) on the proliferation, cell cycle and apoptosis of the GBC-SD and NOZ human GBC cell lines were evaluated by a series of in vitro and in vivo studies. COX-2 and p-ERK1/2 protein expression levels were found to be significantly elevated in GBC tissues as well as in GBC-SD and NOZ cells. Treatments with celecoxib and PD184161 significantly inhibited GBC-SD and NOZ cell growth in a concentration-dependent manner, and their combination produced a synergistic inhibitory effect. In addition, celecoxib and PD184161 significantly inhibited tumor growth in xenograft nude mice. Celecoxib treatment led to G1 arrest via the upregulation of p21 and p27 expression in GBC-SD and NOZ cells, whereas PD184161 did not affect cell cycle distribution. The combination of celecoxib and PD184161 was able to promote cell apoptosis by triggering a collapse of mitochondrial membrane potential and activating caspase-3-mediated apoptosis. In conclusion, COX-2 and p-ERK1/2 protein may serve as potential targets for GBC chemotherapy, and the combination of celecoxib and PD184161 could significantly inhibit GBC cell growth, induce cell G1 arrest and trigger cell apoptosis of GBC cells.

Correction: Long Noncoding RNA GCASPC, a Target of miR-17-3p, Negatively Regulates Pyruvate Carboxylase-Dependent Cell Proliferation in Gallbladder Cancer.

Correction: Long Noncoding RNA GCASPC, a Target of miR-17-3p, Negatively Regulates Pyruvate Carboxylase-Dependent Cell Proliferation in Gallbladder Cancer.

SNORA74B gene silencing inhibits gallbladder cancer cells by inducing PHLPP and suppressing Akt/mTOR signaling

Small nucleolar RNAs (snoRNAs) have been implicated in the development of many cancers. We therefore examined the differential expression of snoRNAs between gallbladder cancer (GBC) tissues and matched adjacent non-tumor tissues using expression microarray analysis with confirmation by quantitative real-time PCR (qRT-PCR). Western blot analysis showed that SNORA74B levels were higher in GBC than non-tumor tissues. SNORA74B expression was positively associated with local invasion, advanced TNM stage, CA19-9 level, and Ki67 expression in patients with GBC, while it was negatively associated with expression of PHLPP, an endogenous Akt inhibitor. Moreover, SNORA74B expression was prognostic for overall survival (OS) and disease-free survival (DFS). Functional studies revealed that silencing SNORA74B in GBC cells using sh-SNORA74B suppressed cell proliferation, induced G1 arrest, and promoted apoptosis. Preliminary molecular investigation revealed that SNORA74B silencing inhibited activation of the AKT/mTOR signaling pathway, while increasing PHLPP expression. PHLPP depletion using shRNA abrogated sh-SNORA74B suppression of GBC cell proliferation, indicating that the antitumor effects of SNORA74B silencing were mediated by PHLPP. These findings define the important role of SNORA74B in cell proliferation, cell cycle, and apoptosis of GBC, and suggest that it may serve as a novel target for GBC treatment.

DUSP1 inhibits cell proliferation, metastasis and invasion and angiogenesis in gallbladder cancer.

DUSP1/MKP1 is a dual-specific phosphatase that regulates MAPK activity and is known to play a key role in tumor biology. Its function in gallbladder cancer (GBC) remains largely unknown, however. By exploring its activities in two GBC cell lines (SGC996 and GBC-SD), DUSP1 was found to inhibit GBC cell proliferation, migration and invasion. Moreover, DUSP1 inhibited GBC growth and metastasis in nude mice subcutaneously xenografted with SGC996 cells. The tumor suppression appeared to be mediated via the DUSP1-pERK/MAPK-MMP2 signal pathway. Angiogenesis was associated with the tumor metastasis in the mouse model and was impaired by DUSP1, which suppressed VEGF expression. These results suggest that DUSP1 suppresses GBC growth and metastasis by targeting the DUSP1-pERK-MMP2/VEGF axis. Identification of the DUSP1-pERK-MMP2/VEGF signals may provide new biomarkers and/or therapeutic targets to better suppress GBC metastasis in the future.

MicroRNA-29c-5p suppresses gallbladder carcinoma progression by directly targeting CPEB4 and inhibiting the MAPK pathway

Gallbladder cancer (GBC) is a leading cause of cancer-related deaths worldwide, and its prognosis remains poor, with a 5-year survival rate of ~5%. Given the crucial role of microRNAs (miRNAs) in cancer metastasis, we aimed to analyze the expression and function of the metastasis-associated miRNA miR-29c-5p in GBC.We validated that expression of miR-29c-5p was significantly downregulated in GBC and was closely associated with lymph node metastasis, overall survival and disease-free survival in 40 GBC patients who were followed clinically. Ectopic overexpression of miR-29c-5p dramatically repressed proliferation, metastasis, and colony formation and induced apoptosis in vitro, and it suppressed tumorigenicity in vivo through the MAPK pathway. Cytoplasmic polyadenylation element binding protein 4 (CPEB4) was identified as a critical effector target of miR-29c-5p. Enforced expression of miR-29c-5p significantly inhibited the expression of CPEB4, and restoration of CPEB4 expression reversed the inhibitory effects of miR-29c-5p on GBC cell proliferation and metastasis. Transforming growth factor-β (TGF-β) upregulated CPEB4 by downregulating miR-29c-5p, leading to MAPK pathway activation. In conclusion, the TGF-β/miR-29c-5p/CPEB4 axis has a pivotal role in the pathogenesis and poor prognosis of GBC, suggesting that miR-29c-5p is a tumor-suppressive miRNA that may serve as potential prognostic biomarker or therapeutic target for GBC.

Casticin induces apoptosis and G0/G1 cell cycle arrest in gallbladder cancer cells

BackgroundCasticin, the flavonoid extracted from Vitex rotundifolia L, exerts various biological effects, including anti-inflammatory and anti-cancer activity. The aim of this study is to investigate the effects and mechanisms of casticin in human gallbladder cancer cells.MethodsHuman NOZ and SGC996 cells were used to perform the experiments. CCK-8 assay and colony formation assay were performed to evaluate cell viability. Cell cycle analyses and annexin V/PI staining assay for apoptosis were measured using flow cytometry. Western blot analysis was used to evaluate the changes in protein expression, and the effect of casticin treatment in vivo was experimented with xenografted tumors.ResultsIn this study, we found that casticin significantly inhibited gallbladder cancer cell proliferation in a dose- and time-dependent manner. Casticin also induced G0/G1 arrest and mitochondrial-related apoptosis by upregulating Bax, cleaved caspase-3, cleaved caspase-9 and cleaved poly ADP-ribose polymerase expression, and by downregulating Bcl-2 expression. Moreover, casticin induced cycle arrest and apoptosis by upregulating p27 and downregulating cyclinD1/cyclin-dependent kinase4 and phosphorylated protein kinase B. In vivo, casticin inhibited tumor growth.ConclusionCasticin induces G0/G1 arrest and apoptosis in gallbladder cancer, suggesting that casticin might represent a novel and effective agent against gallbladder cancer.

MYBL2 is a Potential Prognostic Marker that Promotes Cell Proliferation in Gallbladder Cancer

Background: Gallbladder cancer (GBC) is an aggressive and highly lethal biliary tract malignancy, with extremely poor prognosis. In the present study, we analyzed the potential involvement of MYBL2, a member of the Myb transcription factor family, in the carcinogenesis of human GBC. Methods: MYBL2 expression levels were measured in GBC and cholecystitis tissue specimens using quantitative real-time PCR (qRT-PCR) and immunohistochemical (IHC) assays. The effects of MYBL2 on cell proliferation and DNA synthesis were evaluated using Cell Counting Kit-8 assay (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) retention assay, flow cytometry analysis, western blot, and a xenograft model of GBC cells in nude mice. Results: MYBL2 expression was increased in GBC tissues and associated with histological differentiation, tumour invasion, clinical stage and unfavourable overall survival in GBC patients. The downregulation of MYBL2 expression resulted in the inhibition of GBC cell proliferation, and DNA replication in vitro, and the growth of xenografted tumours in nude mice. Conversely, MYBL2 overexpression resulted in the opposite effects. Conclusions: MYBL2 overexpression promotes GBC cell proliferation through the regulation of the cell cycle at the S and G2/M phase transitions. Thus, MYBL2 could serve as a potential prognostic and therapeutic biomarker in GBC patients.