Investigating Effect of Rapamycin and Metformin on Angiogenesis in Hepatocellular Carcinoma Cell Line

Rastegar, Mandana; Marjani, Haji-Amin; Yazdani, Yaghoub; Shahbazi, Majid; Golalipour, Masoud; Farazmandfar, Touraj

doi:10.15171/apb.2018.008

Cited by 9 publications

(4 citation statements)

References 33 publications

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“…MET was also previously reported to have improved the sensitivity of ovarian cancer cells to MTX, compared with the chemotherapeutic agent MTX alone ( 40 ). Furthermore, another study reported that MET when used in combination with rapamycin decreased cancer cell viability in HepG2 cells by inducing cell apoptosis ( 41 ).…”

Section: Discussionmentioning

confidence: 99%

Effects of Metformin Combined With Antifolates on HepG2 Cell Metabolism and Cellular Proliferation

et al. 2022

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Hepatocellular carcinoma (HCC), one of the most prevalent types of cancers worldwide, continues to maintain high levels of resistance to standard therapy. As clinical data revealed poor response rates, the need for developing new methods has increased to improve the overall wellbeing of patients with HCC. Furthermore, a growing body of evidence shows that cancer metabolic changes are a key feature of many types of human malignancies. Metabolic reprogramming refers to cancer cells’ ability to change their metabolism in order to meet the increased energy demand caused by continuous growth, rapid proliferation, and other neoplastic cell characteristics. For these reasons, metabolic pathways may become new therapeutic and chemopreventive targets. The aim of this study was to investigate the metabolic alterations associated with metformin (MET), an anti-diabetic agent when combined with two antifolate drugs: trimethoprim (TMP) or methotrexate (MTX), and how metabolic changes within the cancer cell may be used to increase cellular death. In this study, single drugs and combinations were investigated using in vitro assays including cytotoxicity assay (MTT), RT-qPCR, annexin V/PI apoptosis assay, scratch wound assay and Seahorse XF analysis, on a human HCC cell line, HepG2. The cytotoxicity assay showed that the IC50 of MET as single therapy was 44.08 mM that was reduced to 22.73 mM and 29.29 mM when combined with TMP and MTX, respectively. The co-treatment of both drugs increased p53 and Bax apoptotic markers, while decreased the anti-apoptotic marker; Bcl-2. Both combinations increased the percentage of apoptotic cells and halted cancer cell migration when compared to MET alone. Furthermore, both combinations decreased the MET-induced increase in glycolysis, while also inducing mitochondrial damage, altering cancer cell bioenergetics. These findings provide an exciting insight into the anti-proliferative and apoptotic effects of MET and anti-folates on HepG2 cells, and how in combination, may potentially combat the aggressiveness of HCC.

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Section: Discussionmentioning

confidence: 99%

Effects of Metformin Combined With Antifolates on HepG2 Cell Metabolism and Cellular Proliferation

et al. 2022

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“…With the use of the drug, though, a considerable portion of HCC was found resistant to sorafenib, even multiple-drug resistant [ 29 , 30 ]. The main strategy for overcoming the resistance to chemotherapy is combination therapy; that is, two drugs that inhibit one or more key signal pathways in different ways, thus, blocking resistance mechanisms [ 31 – 33 ]. There are many mechanisms of acquired sorafenib resistance in HCC cells, of which abnormal activation of the PI3K/AKT/mTOR pathway plays an important role [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

BEZ235 Increases the Sensitivity of Hepatocellular Carcinoma to Sorafenib by Inhibiting PI3K/AKT/mTOR and Inducing Autophagy

Cao

Liu

Zhang

et al. 2021

BioMed Research International

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Acquired resistance of hepatocellular carcinoma (HCC) to sorafenib (SFB) is the main reason for the failure of SFB treatment of the cancer. Abnormal activation of the PI3K/AKT/mTOR pathway is important in the acquired resistance of SFB. Therefore, we investigated whether BEZ235 (BEZ) could reverse acquired sorafenib resistance by targeting the PI3K/mTOR pathway. A sorafenib-resistant HCC cell line Huh7R was established. MTT assay, clone formation assay, flow cytometry, and immunofluorescence were used to analyze the effects of BEZ235 alone or combined with sorafenib on cell proliferation, cell cycle, apoptosis, and autophagy of Huh7 and Huh7R cells. The antitumor effect was evaluated in animal models of Huh7R xenografts in vivo. Western blot was used to detect protein levels of the PI3K/AKT/mTOR pathway and related effector molecules. In vitro results showed that the Huh7R had a stronger proliferation ability and antiapoptosis effect than did Huh7, and sorafenib had no inhibitory effect on Huh7R. SFB + BEZ inhibited the activation of the PI3K/AKT/mTOR pathway caused by sorafenib. Moreover, SFB + BEZ inhibited the proliferation and cloning ability, blocked the cell cycle in the G0/G1 phase, and promoted apoptosis in the two cell lines. The autophagy level in Huh7R cells was higher than in Huh7 cells, and BEZ or SFB + BEZ further promoted autophagy in the two cell lines. In vivo, SFB + BEZ inhibited tumor growth by inducing apoptosis and autophagy. We concluded that BEZ235 enhanced the sensitivity of sorafenib through suppressing the PI3K/AKT/mTOR pathway and inducing autophagy. These observations may provide the experimental basis for sorafenib combined with BEZ235 in trial treatment of HCC.

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“…The differences in response to rapamycin and metformin treatment have been previously demonstrated. For example, rapamycin but not metformin decreased expression of vascular endothelial growth factor (VEGF) in hepatocellular carcinoma cells 46 . Furthermore, rapamycin treatment inhibited the growth of hepatocarcinoma tumors inoculated in NOD-SCID mice to a greater extent as compared to metformin 47 .…”

Section: Discussionmentioning

confidence: 99%

Pharmacological inhibition of mTORC1 increases CCKBR-specific tumor uptake of radiolabeled minigastrin analogue [¹⁷⁷Lu]Lu-PP-F11N

et al. 2020

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Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [ 177 Lu]Lu-DOTA-(DGlu) 6 -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 ([ 177 Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers. Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [ 177 Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation. Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [ 177 Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [ 177 Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of β-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [ 177 Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [ 177 Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment. Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [ 177 Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the lev...

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Investigating Effect of Rapamycin and Metformin on Angiogenesis in Hepatocellular Carcinoma Cell Line

Cited by 9 publications

References 33 publications

Effects of Metformin Combined With Antifolates on HepG2 Cell Metabolism and Cellular Proliferation

Effects of Metformin Combined With Antifolates on HepG2 Cell Metabolism and Cellular Proliferation

BEZ235 Increases the Sensitivity of Hepatocellular Carcinoma to Sorafenib by Inhibiting PI3K/AKT/mTOR and Inducing Autophagy

Pharmacological inhibition of mTORC1 increases CCKBR-specific tumor uptake of radiolabeled minigastrin analogue [¹⁷⁷Lu]Lu-PP-F11N

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Investigating Effect of Rapamycin and Metformin on Angiogenesis in Hepatocellular Carcinoma Cell Line

Cited by 9 publications

References 33 publications

Effects of Metformin Combined With Antifolates on HepG2 Cell Metabolism and Cellular Proliferation

Effects of Metformin Combined With Antifolates on HepG2 Cell Metabolism and Cellular Proliferation

BEZ235 Increases the Sensitivity of Hepatocellular Carcinoma to Sorafenib by Inhibiting PI3K/AKT/mTOR and Inducing Autophagy

Pharmacological inhibition of mTORC1 increases CCKBR-specific tumor uptake of radiolabeled minigastrin analogue [177Lu]Lu-PP-F11N

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Pharmacological inhibition of mTORC1 increases CCKBR-specific tumor uptake of radiolabeled minigastrin analogue [¹⁷⁷Lu]Lu-PP-F11N