Lentiviral-Mediated Beclin-1 Overexpression Inhibits Cell Proliferation and Induces Autophagy of Human Esophageal Carcinoma Eca109 Cell Xenograft in Nude Mice

2020 ◽  
Vol 15 (1) ◽  
pp. 70-77
Author(s):  
Junhe Zhang ◽  
Weihua Dong
Keyword(s):  
Growth Rate ◽  
Cell Proliferation ◽  
Esophageal Carcinoma ◽  
Nude Mice ◽  
Beclin 1 ◽  
Control Group ◽  
Vector Group ◽  
Empty Vector ◽  
Empty Vector Group

Background: Esophageal carcinoma is one of the common malignant tumors in digestive tract. BECLIN-1 is a key gene that regulates autophagy, and its abnormal expression may be related with many human tumors. However, the mechanism of BECLIN-1 in esophageal carcinoma remains unknown. Objective: In this study, we explored the effect of BECLIN-1 overexpression on tumor growth in mice with esophageal carcinoma and its mechanism. Methods: Recombined lentiviral vector containing BECLIN-1 was used to transfect human esophageal carcinoma Eca109 cells and establish stable cell line. qRT-PCR was used to detect BECLIN-1 mRNA level in the transfected Eca109 cells, CCK-8 assay was used to detect cell proliferation. Beclin-1, P62 and LC3-II protein expression levels in Eca109 cells were detected using Western blot analysis. Subcutaneous xenograft nude mice model of human esophageal carcinoma was established, and the tumor growths in Beclin-1 group, control group and empty vector group were monitored. Beclin-1 protein expression in vivo was detected by immunohistochemistry. Results: Beclin-1 mRNA and protein were overexpressed in Eca109 cells. Compared with empty vector group, the growth rate of cells transfected with BECLIN-1 decreased significantly. Compared with the control group and empty vector group, the expression level of P62 protein in beclin-1 group was significantly decreased, while the expression level of LC3-II protein was significantly increased. The tumor growth rate in nude mice of Beclin-1 group was significantly lower than that of the control group and empty vector group, and Beclin-1 protein was mainly expressed in Beclin-1 group in vivo. Conclusion: BECLIN-1 can induce autophagy in esophageal carcinoma Eca109 cells, and it can significantly inhibit the growth of esophageal carcinoma.

scholarly journals LncRNA H19 as a Competing Endogenous RNA to Regulate AQP Expression in the Intestinal Barrier of IBS-D Patients

2021 ◽  
Vol 11 ◽  
Author(s):  
Guanqun Chao ◽  
Zhaojun Wang ◽  
Yi Yang ◽  
Shuo Zhang
Keyword(s):  
Intestinal Barrier ◽  
Control Group ◽  
Vector Group ◽  
Empty Vector ◽  
Non Coding Rna ◽  
Irritable Bowel ◽  
Long Non Coding Rna ◽  
Empty Vector Group

ObjectiveThe study aimed to investigate the role of Long non-coding RNA (LncRNA) H19 in the pathogenesis of Diarrhea Irritable Bowel Syndrome (IBS-D), and further to the regulatory effect of LncRNA H19 on AQP1, 3 in the intestinal mucosa of IBS-D patients, so as to seek a new way to elucidate the mechanism of IBS in clinic.MethodsThe levels of LncRNA H19, AQP1, and AQP3 were detected in colonic tissues of IBS-D patients, compared with that in healthy controls. Through RNA gene interference and activation methods, small activating RNA (saRNA) and small interfering (siRNA) were transfered into Caco-2 cells in vitro experiment, and sub-group for two control group, siH19 empty vector group, siH19 interference group, overexpression H19 vector group, and overexpression H19 empty vector group. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot were applied to evaluate the expression levels of LncRNA H19 and the amount of AQP1 and AQP3 protein expression, respectively.ResultsCompared with healthy volunteers, the levels of LncRNA H19, AQP1, and AQP3 in the colonic mucosa of IBS-D patients were significantly decreased (P < 0.05). The results in vitro transfection experiment revealed that the level of LncRNA H19 in the siH19 interference group was significantly declined (P < 0.05), while there was a remarkable increase in the overexpression H19 vector group (P < 0.05), compared with the corresponding control groups. The expression of AQP1 and AQP3 in Caco-2 cells was of positive correlation with the level of LncRNA H19.ConclusionThat the down-regulation of LncRNA H19 resulted in the expression changes of AQP1 and AQP3 may play an important role in the occurrence and development of IBS-D.

scholarly journals Obesity Potentiates Esophageal Squamous Cell Carcinoma Growth and Invasion by AMPK-YAP Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Jia-Huang Liu ◽  
Qi-Fei Wu ◽  
Jun-Ke Fu ◽  
Xiang-Ming Che ◽  
Hai-Jun Li
Keyword(s):  
Cell Proliferation ◽  
Squamous Cell Carcinoma ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Nude Mice ◽  
Serum Glucose ◽  
Migration And Invasion ◽  
Endocrine Effect

Obesity could increase the risk of esophageal squamous cell carcinoma (ESCC) and affect its growth and progression, but the mechanical links are unclear. The objective of the study was to explore the impact of obesity on ESCC growth and progression utilizing in vivo trials and cell experiments in vitro. Diet-induced obese and lean nude mice were inoculated with TE-1 cells, then studied for 4 weeks. Serum glucose, insulin, leptin, and visfatin levels were assayed. Sera of nude mice were obtained and then utilized to culture TE-1. MTT, migration and invasion assays, RT-PCR, and Western blotting were used to analyze endocrine effect of obesity on cell proliferation, migration, invasion, and related genes expression of TE-1. Obese nude mice bore larger tumor xenografts than lean animals, and were hyperglycemic and hyperinsulinemic with an elevated level of leptin and visfatin in sera, and also were accompanied by a fatty liver. As for the subcutaneous tumor xenograft model, tumors were more aggressive in obese nude mice than lean animals. Tumor weight correlated positively with mouse body weight, liver weight of mice, serum glucose, HOMA-IR, leptin, and visfatin. Obesity prompted significant TE-1 cell proliferation, migration, and invasion by endocrine mechanisms and impacted target genes. The expression of AMPK and p-AMPK protein decreased significantly ( P < 0.05 ); MMP9, total YAP, p-YAP, and nonphosphorylated YAP protein increased significantly ( P < 0.05 ) in the cells cultured with conditioned media and xenograft tumor from the obese group; the mRNA expression of AMPK decreased significantly ( P < 0.05 ); YAP and MMP9 mRNA expression increased significantly ( P < 0.05 ) in the cells exposed to conditioned media from the obese group. In conclusion, the altered adipokine milieu and metabolites in the context of obesity may promote ESCC growth in vivo; affect proliferation, migration, and invasion of ESCC cells in vitro; and regulate MMP9 and AMPK-YAP signaling pathway through complex effects including the endocrine effect.

scholarly journals p62 Overexpression Promotes Bone Metastasis of Lung Adenocarcinoma out of LC3-Dependent Autophagy

2021 ◽  
Vol 11 ◽  
Author(s):  
Dongqi Li ◽  
Chuanchun He ◽  
Fan Ye ◽  
En Ye ◽  
Hao He ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Overall Survival ◽  
Bone Metastasis ◽  
Lung Adenocarcinoma ◽  
Survival Rates ◽  
A549 Cells ◽  
Adaptor Protein ◽  
Beclin 1 ◽  
Cox Regression Analysis

p62 protein has been implicated in bone metastasis and is a multifunctional adaptor protein usually correlated with autophagy. Herein, we investigated p62 expression and its prognostic significance in bone metastasis of lung adenocarcinoma, and analyzed whether the mechanism involved depends on autophagy. mRNA and protein expression of p62, LC3B and Beclin 1 were detected by reverse transcription-quantitative PCR and western blotting, respectively, in fresh bone metastasis tissues (n=6 cases) and normal cancellous bone tissues (n=3 cases). The association between p62 and LC3B expression and patient prognosis was subsequently analyzed in 62 paraffin-embedded bone metastasis specimens by immunohistochemistry assay. Small interfering RNA (siRNA) was employed to downregulate p62 expression in SPC-A-1 and A549 cells. Cell proliferation and migration ability were tested by CCK8, CCF and Transwell assays respectively. Autophagy was induced by Rapamycin or inhibited by Atg 7 knockout/Chloroquine in A549 cells and p62 and LC3II/I expression were analyzed. After subcutaneous inoculation or intracardial injection of A549 cells into nude mice, the effect of p62 downregulation in vivo was analyzed by histopathological examination. The results showed that p62, LC3B and Beclin 1 mRNA and protein were all overexpressed in bone metastasis tissues (all P&lt;0.01). Patient samples with high p62 expression levels were significantly associated with more bone lesions (&gt;3), shorter overall survival rates and shorter progression free survival rates compared with patients having lower p62 expression (P=0.014, P=0.003, P=0.048, respectively). Cox regression analysis identified p62 expression as an independent prognostic indicator of overall survival of patients with bone metastasis (P=0.007). In vitro p62 downregulation inhibited SPC-A-1 and A549 cells migration but had no effect on cell proliferation. After autophagy induction or inhibition, p62 expression involved in autophagy flux and changed inconsistently according to the switch of LC3I to LC3II in different autophagy conditions. In vivo p62 downregulation had no effect on growth of subcutaneous tumor. Lung or bone metastasis lesion was not found in all mice model. These findings suggested that p62 overexpression promotes tumor cell invasion out of LC3-dependent autophagy, which could be used a potential prognostic biomarker and therapeutic target for bone metastasis of lung adenocarcinoma.

scholarly journals Exploiting GRK2 Inhibition as a Therapeutic Option in Experimental Cancer Treatment: Role of p53-Induced Mitochondrial Apoptosis

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3530
Author(s):  
Jessica Gambardella ◽  
Antonella Fiordelisi ◽  
Gaetano Santulli ◽  
Michele Ciccarelli ◽  
Federica Andrea Cerasuolo ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cancer Cell ◽  
Nude Mice ◽  
Specific Inhibitor ◽  
Cancer Cell Proliferation ◽  
In Vivo Models ◽  
P53 Expression

The involvement of GRK2 in cancer cell proliferation and its counter-regulation of p53 have been suggested in breast cancer even if the underlying mechanism has not yet been elucidated. Furthermore, the possibility to pharmacologically inhibit GRK2 to delay cancer cell proliferation has never been explored. We investigated this possibility by setting up a study that combined in vitro and in vivo models to underpin the crosstalk between GRK2 and p53. To reach this aim, we took advantage of the different expression of p53 in cell lines of thyroid cancer (BHT 101 expressing p53 and FRO cells, which are p53-null) in which we overexpressed or silenced GRK2. The pharmacological inhibition of GRK2 was achieved using the specific inhibitor KRX-C7. The in vivo study was performed in Balb/c nude mice, where we treated BHT-101 or FRO-derived tumors with KRX-C7. In our in vitro model, FRO cells were unaffected by GRK2 expression levels, whereas BHT-101 cells were sensitive, thus suggesting a role for p53. The regulation of p53 by GRK2 is due to phosphorylative events in Thr-55, which induce the degradation of p53. In BHT-101 cells, the pharmacologic inhibition of GRK2 by KRX-C7 increased p53 levels and activated apoptosis through the mitochondrial release of cytochrome c. These KRX-C7-mediated events were also confirmed in cancer allograft models in nude mice. In conclusion, our data showed that GRK2 counter-regulates p53 expression in cancer cells through a kinase-dependent activity. Our results further corroborate the anti-proliferative role of GRK2 inhibitors in p53-sensitive tumors and propose GRK2 as a therapeutic target in selected cancers.

Foxp3 in Lewis Lung Cancer Cells Enhances the Expression of TGF-β1 and IL-10

2014 ◽  
Vol 998-999 ◽  
pp. 252-255
Author(s):  
Li Song Chen ◽  
Xiao Dong Gai ◽  
Chun Li
Keyword(s):  
Lung Cancer ◽  
Immune Escape ◽  
Lewis Lung ◽  
Rt Pcr ◽  
Vector Group ◽  
Lewis Lung Cancer ◽  
Tumor Immune Escape ◽  
Empty Vector ◽  
Tgf Β1 ◽  
Empty Vector Group

In order to explore the related mechanism of Foxp3 in tumor immune escape, the study detected the expression of Foxp3 in lewis lung cancer (LLC) cells and analyzed the expression of TGF-β1 and IL-10 in Foxp3 overexpressed LLC cells. Foxp3 mRNA was detected in LLC cells by RT-PCR. Foxp3 was highly expressed in Foxp3 transfected LLC group than that of in empty vector group and LLC group by RT-PCR(P <0.01). The mRNA and protein expression of TGF-β1 and IL-10 significantly increased in Foxp3 transfected LLC group than that of in empty vector group and LLC group by RT-PCR and ELISA(P <0.05). These results suggest that Foxp3 in LLC cells may promote tumor immune escape by enhancing the expression of TGF-β1 and IL-10.

scholarly journals Treatment with the Recombinant SLIT2 Protein Delays AML Leukemogenesis In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2368-2368
Author(s):  
Luise A de Albuquerque Simoes ◽  
Isabel Weinhäuser ◽  
Diego A Pereira-Martins ◽  
César Alexander Ortiz Rojas ◽  
Thiago Mantello Bianco ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Progression ◽  
Functional Role ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Leukemic Cells ◽  
Control Group

Abstract Accumulating evidence suggest that the axon guidance molecules SLIT and ROBO are not only implicated in physiological process but also in cancer progression. Depending on the type of cancer the SLIT-ROBO axis can either act as a tumor suppressor gene, in which case the SLIT2 promoter site is frequently hypermethylated or as an oncogene, whereby high expression is often associated with poor prognosis. In the context of acute myeloid leukemia (AML), low expression of SLIT2 has been associated with low overall survival (OS) (Golos et al., 2019), while the functional role of SLIT2 remains largely unknown. Recently, we showed that the knockdown of SLIT2 increased cell proliferation of acute promyelocytic leukemia (APL) cells resulting in a more aggressive course of disease progression in vivo using the murine transgenic APL model (Weinhäuser et al., 2020). Here, we aimed to study the functional role of SLIT2 in a more heterogeneous disease, such as AML. Using different publicly available datasets. (GSE58477, normal karyotype blasts: 62, healthy CD34 +: 10; GSE63409, LSC: 14, HSC: 5) we detected increased methylation at the SLIT2 promoter site of AML leukemic cells compared to healthy CD34 + cells suggesting SLIT2 tumor suppressive functions. In addition, we measured decreased levels of SLIT2 in the bone marrow (BM) plasma of AML patients compared to healthy donors. To assess the biological role of SLIT2, we treated AML cell lines (KASUMI1, MV411, and MOLM13) with recombinant SLIT2 (50 ng/mL) in vitro. Administration of SLIT2 reduced AML cell growth, colony formation and induced cell cycle arrest in the G1 phase for all AML cell lines. Conversely, the knockdown of SLIT2 promoted increased THP-1 and OCI-AML3 cell proliferation. Next, we determined whether the treatment with SLIT2 could delay leukemogenesis in vivo using the AML cell line MV4-11. Engraftment was monitored by luciferase bioluminescent signal and NSGS mice were either treated with recombinant SLIT2 using a dose of 25 ng/g of body weight or vehicle (control group). SLIT2 therapy resulted in a lower disease burden, decreased leukemic infiltration in the BM and spleen, reduced spleen size, and increased OS compared to the control group (p&lt;0.05). In conclusion, we showed that SLIT2 methylation is recurrent in AML patients and that the level of SLIT2 in the plasma of AML patients is reduced. Moreover, SLIT2 treatment appears to have a cytostatic effect on different AML cell lines delaying leukemogenesis in vivo. Overall, our study reveals the therapeutic potential of SLIT2 in hematological malignancies, which could be used as an adjuvant in the clinic. Disclosures No relevant conflicts of interest to declare.

scholarly journals Plasmodium circumsporozoite protein enhances the efficacy of gefitinib in lung adenocarcinoma cells by inhibiting autophagy via proteasomal degradation of LC3B

2021 ◽  
Author(s):  
Xiao Lu ◽  
Jiao Zhang ◽  
Quanxing Liu ◽  
Dong Zhou ◽  
Xufeng Deng ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Viability ◽  
Nude Mice ◽  
Specific Inhibitor ◽  
Proteasomal Degradation ◽  
Circumsporozoite Protein ◽  
Control Group ◽  
Egfr Tkis

Abstract Background: Almost all patients with lung adenocarcinoma (LUAD) develop resistance to EGFR-TKIs, which limit the long-term clinical application of these agents. Accumulating evidence shows one of the main reasons for resistance to EGFR-TKIs is induction of autophagy in tumor cells. Our previous study found that circumsporozoite protein (CSP) in Plasmodium can suppress autophagy in host hepatocytes. However, it is unknown whether CSP-mediated inhibition of autophagy could improve the anti-tumor effect of EGFR-TKIs. Methods: We constructed A549 and H1975 cell lines with stable overexpression of CSP (OE-CSP cells). CCK-8, LDH, flow cytometry, and colony analysis were performed to observe the effect of CSP overexpression on cell viability, the apoptosis rate, and stemness. The sensitizing effect of CSP on gefitinib was evaluated in vivo using a subcutaneous tumor model in nude mice and immunohistochemical assay. The role of CSP in regulation of autophagy was investigated by laser confocal microscopy assay and Western blotting. A transcriptome sequencing assay and real-time polymerase chain reaction were used to determine the levels of mRNA for autophagy-related proteins. Cyclohexane, MG132, TAK-243, and immunoprecipitation assays were used to detect and confirm proteasomal degradation of LC3B. Results: OE-CSP A549 and H1975 cells were more sensitive to gefitinib, demonstrating significant amounts of apoptosis and decreased viability. In the OE-CSP group, autophagy was significantly inhibited, and there was a decrease in LC3B protein after exposure to gefitinib. Cell viability and stemness were recovered when OE-CSP cells were exposed to rapamycin. In nude mice with xenografts of LUAD cells, inhibition of autophagy by CSP resulted in suppression of cell growth and more marked apoptosis during exposure to gefitinib. CSP promoted ubiquitin-proteasome degradation of LC3B, leading to inhibition of autophagy in LUAD cells after treatment with gefitinib . When LUAD cells were treated with the ubiquitin-specific inhibitor TAK-243, cell viability, apoptosis, and growth were comparable between the OE-CSP group and a control group both in vivo and in vitro . Conclusions: CSP can inhibit gefitinib-induced autophagy via proteasomal degradation of LC3B, which suggests that CSP could be used as an autophagy inhibitor to sensitize EGFR-TKIs.

Evaluation of Anti-Tumor Potential of Lactobacillus acidophilus ATCC4356 Culture Supernatants in MCF-7 Breast Cancer

2020 ◽  
Vol 21 ◽  
Author(s):  
Ramazan Behzadi ◽  
Ahmad Hormati ◽  
Karim Eivaziatashbeik ◽  
Sajjad Ahmadpour ◽  
Fatemeh Khodadust ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Nude Mice ◽  
Control Group ◽  
Bacterial Strains ◽  
Anti Cancer ◽  
99Mtc Mibi ◽  
Culture Supernatants ◽  
Mcf 7

Background: Anti-cancer activity of some lactic acid bacterial strains is well documented in several literatures. Lactobacillus strains have received considerable attention as a beneficial microbiota. The aim of this study is to evaluate the effects of anti-tumor activities of L. acidophilus ATCC4356 culture supernatants on the MCF-7 human breast cancer cells. Materials and methods: Anti-cancer effect of 24h and 48h culture supernatants at various concentrations (1.25, 2.5, 5, 10 and 20 µg/ml) were determined by various in vitro and in vivo assays including MTT, tumor volume measurement as well as 99mTc-MIBI biodistribution in MCF-7 tumor bearing nude mice and histopathology test. For evaluation of the related mechanism of action, quantitative PCR was conducted. Results: The 48h culture supernatants at 10 and 20 µg/ml exhibited significant in vitro inhibition of MCF-7 cell proliferation. However, this inhibition was not observed for HUVEC human endothelial normal cells. Q-PCR indicated that treatment by the supernatant led to a significant downregulation of VEGFR ( ̴ 0.009 fold) and Bcl-2 ( ̴ 0.5 fold) and upregulation of p53 ( ̴ 1.3 fold). In vivo study using MCF-7 xenograft mouse models demonstrated reduction in tumor weight and volume by both 24h and 48h supernatants (10 µg/ml and 20 µg/ml) after 15 days. According to the 99mTc-MIBI biodistribution result, treatment of MCF-7 bearing nude mice with both 24h and 48h supernatant (20 µg/ml) led to significant decrease in tumor uptake compared with the control group. Conclusion: These results suggest that the culture supernatants of L. acidophilus ATCC4356 at suitable concentrations can be considered as a good alternative nutraceutical with promising therapeutic indexes for breast cancer.

Octreotide Reduces Pancreatic Islet Apoptosis and Improves Islet Transplantation Efficiency In Vitro and In Vivo

2020 ◽  
Vol 16 (7) ◽  
pp. 1082-1101
Author(s):  
Biao Yang ◽  
Yiming Zhou ◽  
Lei Tian ◽  
Yunfei Lu
Keyword(s):  
Cell Proliferation ◽  
Islet Transplantation ◽  
Islet Cell ◽  
Control Group ◽  
Average Cell ◽  
Min6 Cells ◽  
Hypoxic Conditions ◽  
Octreotide Treatment

The drug octreotide, a somatostatin analog, stimulates the cellular free radical scavenging system and inhibits the release of superoxide anions from monocytes. We hypothesized that octreotide also protects islet β cell function and improves the survival of transplanted islets by ameliorating the adverse effects of hypoxia and reoxygenation on these cells, thus inhibiting apoptosis. To test this hypothesis, we experimentally induced hypoxia in islet cells in mouse insulinoma Min6 cells. Octreotide treatment mildly but significantly improved cell viability under normoxic and hypoxic conditions. Secreted vascular endothelial growth factor (VEGF) from the Min6 cells was downregulated after octreotide treatment during hypoxia. By contrast, the expression of hypoxia-inducible factor (HIF)-1α was upregulated after octreotide treatment under both normoxic and hypoxic conditions. Octreotide treatment also lowered the apoptotic rate of Min6 cells under hypoxic conditions in vitro. In a mouse transplant model, octreotide improved the post-transplantation efficacy and function of islet grafts. Expression of p53 and Bax in islet grafts was upregulated in the recipients treated with octreotide one day after islet transplantation, and the octreotide-treated group produced significantly less Bax than the control group on days 3 and 7 following transplantation. TUNEL assay further demonstrated a decrease in islet cell apoptosis in the octreotide group on days 1, 3, 7, and 14 after transplantation compared with that of the control group (P < 0.05). No islet cell proliferation was found in the octreotide and control groups on days 1, 3, and 7 following transplantation. However, by day 14, the group treated with octreotide demonstrated significantly higher average cell proliferation rates than the controls did (P < 0.05). Thus, octreotide decreased the apoptosis of islets under hypoxic conditions in vitro and enhanced the efficacy of islet transplantation in vivo. Octreotide has excellent potential for therapeutic applications in islet transplantation and merits further study.

scholarly journals The small molecule kobusone can stimulate islet β-cell replication in vivo

2021 ◽  
Vol 49 (7) ◽  
pp. 030006052110328
Author(s):  
Jin woo Choi ◽  
Jin-deok Joo ◽  
Jang hyeok In ◽  
Daewoo Kim ◽  
Yongshin Kim ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Body Weight ◽  
Serum Insulin ◽  
Cyclin D3 ◽  
Control Group ◽  
Cell Replication ◽  
Β Cell ◽  
Insulin Levels ◽  
Glucose Levels

Objective To investigate the ability of kobusone to reduce high glucose levels and promote β-cell proliferation. Methods Four-week-old female db/db mice were assigned to the kobusone (25 mg/kg body weight, intraperitoneally twice a day) or control group (same volume of PBS). Glucose levels and body weight were measured twice a week. After 6 weeks, intraperitoneal glucose tolerance tests and immunohistochemical studies were performed, and insulin levels were determined. The expression of mRNAs involved in cell proliferation, such as PI3K, Akt, cyclin D3 and p57Kip 2 , was measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results Kobusone reduced blood glucose levels after 3 weeks and more strongly increased serum insulin levels than the vehicle. Immunohistochemistry illustrated that kobusone increased 5-bromo-2′-deoxyuridine incorporation into islet β-cells, suggesting that it can stimulate islet β-cell replication in vivo. RT-qPCR indicated that kobusone upregulated the mRNA expression of PI3K, Akt, and cyclin D3 and downregulated that of p57Kip2. Conclusion Our findings suggest that kobusone is a potent pancreatic islet β-cell inducer that has the potential to be developed as an anti-diabetic agent.

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