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Objective: To explore whether metformin (MET) can affect the biological behaviour and CD133 mRNA expression of CD133 + colon cancer stem cells (CCSCs) through miR-342-3p., Methods: The direct immunomagnetic bead method was used to select CD133 + CCSCs from the SW480 and HCT116 cell lines, and miRNA-tailing qRT-PCR was used to detect the expression changes of tumor suppressor-related miRNAs (miR-34a, miR-126, miR-143, miR-145, miR-342-3p, miR-342-5p) after MET intervention. Then, miR-342-3p with markedly significant differential expression was selected as the target miRNA. The lentiviruses LV16-hsa-miR-342-3p inhibitor and LV16-NC were used for the transfection inhibition test. CCK-8, flow cytometry, and qRT-PCR were used to detect the cell viability, apoptosis rate, and CD133 mRNA expression of CD133 + CCSCs., Results: Under the high-glucose environment, the expression of tumor suppressor-related miRNAs in CCSCs changed differently ( p <0.05), MET also had different effects on the expression of tumor suppressor-related miRNA under different glucose concentrations ( p <0.05). Among them, MET upregulates the expression of miR-342-3p in CCSCs for the first time. The results of the lentiviruses transfection inhibition test showed that after miR-342-3p was inhibited, the cell viability and apoptosis rate of CD133 + CCSCs did not change significantly compared with before inhibition ( p >0.05), but the expression of CD133 mRNA markedly increased ( p <0.05). Meanwhile, after MET intervention, the apoptosis rate and the expression of CD133 mRNA of CD133 + CCSCs was significantly increased, and the proliferation of CD133 + CCSCs was obviously inhibited ( p <0.05)., Conclusion: MET upregulating the expression of miR-342-3p may not have a significant effect on the proliferation and apoptosis of CD133 + CCSCs, but it can reduce the expression of CD133 mRNA in CD133 + CCSCs., Competing Interests: The authors declare no conflict of interest., (© 2021 Zhang et al.)

Despite significant advances in therapy, the 5-year survival rates for patients with advanced stage oral cancers still remains poor as an appropriate treatment has not been found yet, due to side effects of chemo/radiotherapy. Verbascoside (VB), a major bioactive constituent of the Tsoong herb, displays pharmacological properties by exhibiting anti-oxidative, anti-inflammatory and anti-cancer activities. However, the underlining function and mechanism of VB in human oral squamous cell carcinoma (OSCC) remains unclear. In this study, we show that VB significantly decreased the viability and metastasis of HN4 and HN6 tumor cells, while promoting apoptosis. A xenograft OSCC mouse model further showed that intraperitoneal injection of VB strongly inhibited growth and lung metastasis of implanted tumor cells. Immunoblot analysis confirmed that VB effectively suppressed nuclear factor (NF)-κB activation and downstream Bcl-2/Bcl-XL expression, resulting in increased OSCC cell apoptosis. In addition, VB suppressed mRNA and protein expression of matrix metalloproteinase-9 via suppression of NF-κB activation, thereby inhibiting tumor cell metastasis. Inspiringly, compared to cisplatin-treated group, VB is a biocompatible agent without signficant side effects in vivo. Collectively, our results demonstrate that VB effectively inhibits OSCC tumor cell growth and metastasis via suppression of IκB kinase complex (IKK)/NF-κB-related signaling activation, suggesting that VB has potential use as a potent anticancer agent in OSCC therapeutic strategies., (© 2018 UICC.)

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists.

TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through its death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. The selectivity of TRAIL towards cancer cells has promoted clinical evaluation of recombinant human TRAIL (rhTRAIL) and its agonistic antibodies in treating several major human cancers including colon and non-Hodgkin's lymphoma. However, little is known about their ability in killing oral squamous cell carcinoma (OSCC) cells. In this study, we tested the apoptotic responses of a panel of seven human OSCC cell lines (HN31, HN30, HN12, HN6, HN4, Cal27, and OSCC3) to rhTRAIL and monoclonal antibodies against DR4 or DR5. We found that rhTRAIL is a potent inducer of apoptosis in most of the oral cancer cell lines tested both in vitro and in vivo. We also showed that DR5 was expressed on the surface of the tested cell lines which correlated with the cellular susceptibility to apoptosis induced by rhTRAIL and anti-DR5 antibody. By contrast, little or no DR4 was detected on the surface of OSCC3 and HN6 cells rendering cellular resistance to DR4 antibody and a reduced sensitivity to rhTRAIL. Notably, the overall TRAIL sensitivity correlated well with the levels of endogenous active Ras in the cell lines tested. Expression of a constitutively active Ras mutant (RasV12) in OSCC3 cells selectively upregulated surface expression of DR5, but not DR4, and restored TRAIL sensitivity. Our findings could have implications for the use of TRAIL receptor targeted therapies in the treatment of human OSCC tumors particularly the ones harboring constitutively active Ras mutant.

Rac1 GTPase regulates a variety of signaling pathways that are implicated in malignant phenotypes. Here, we show that selective inhibition of Rac1 activity by the pharmacologic inhibitor NSC23766 suppressed cell growth in a panel of human breast cancer cell lines, whereas it had little toxicity to normal mammary epithelial cells. NSC23766 elicits its cytotoxicity via two distinct mechanisms in a cell line-dependent manner: induction of G(1) cell cycle arrest in cell lines (MDA-MB-231, MCF7, and T47D) that express retinoblastoma (Rb) protein or apoptosis in Rb-deficient MDA-MB-468 cells. In MDA-MB-231 cells, Rac1 inhibition induced G(1) cell cycle arrest through downregulation of cyclin D1 and subsequent dephosphorylation/inactivation of Rb. By contrast, MDA-MB-468 cells underwent substantial apoptosis that was associated with loss of antiapoptotic proteins survivin and X-linked inhibitor of apoptosis protein (XIAP). Rac1 knockdown by RNAi interference confirmed the specificity of NSC23766 and requirement for Rac1 in the regulation of cyclin D1, survivin, and XIAP in breast cancer cells. Further, NF-kappaB, but not c-Jun NH(2)-terminal kinase or p38 pathways, mediates the survival signal from Rac1. Overall, our results indicate that Rac1 plays a central role in breast cancer cell survival through regulation of NF-kappaB-dependent gene products.

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is being evaluated clinically in treating various malignancies. Previous studies have shown that repeated application of high doses of rhTRAIL results in a subpopulation of parental cells that is unresponsive to the death ligand. However, it is not clear whether TRAIL-sensitive cancer cells could acquire resistance to TRAIL treatment. Here, we found that MDA-MB-231 breast cancer cells, which are highly sensitive to TRAIL-induced apoptosis, became resistant to TRAIL killing after a prolonged exposure to subtoxic doses of rhTRAIL. The resulting TRAIL-resistant cells were cross-resistant to antibodies against its death receptors (DR4 and DR5); however, they retained sensitivity to several clinically relevant chemotherapies. Surface expression of DR4 and DR5 was significantly reduced in the selected cells, resulting in failure in death-inducing signaling complex formation and caspase activation. In addition, real-time PCR analysis revealed an upregulation in multiple apoptosis-regulator genes, including c-FLIP, Stat5a, and Stat5b. Inhibition of Janus-activated kinase, an upstream activator of signal transducer and activator of transcription 5 (Stat5), or knockdown of Stat5 itself partially restored cellular sensitivity to TRAIL-induced apoptosis, suggesting that Stat5 signaling is also involved in the development of TRAIL resistance. Furthermore, we showed that acquired TRAIL resistance was effectively eliminated by combination with etoposide, doxorubicin, or paclitaxel. These results suggest that tumor cells could acquire resistance to TRAIL therapy especially when they are repeatedly exposed to low levels of the death ligand, highlighting the necessity of combination with therapies that target the resistance mechanisms.

Physiological oxidants that are generated by activated phagocytes comprise the main source of oxidative stress during inflammation. Oxidants such as taurine chloramine (TnCl) and hydrogen peroxide (H(2)O(2)) can damage proteins and induce apoptosis, but the role of specific protein oxidation in this process has not been defined. We found that the actin-binding protein cofilin is a key target of oxidation. When oxidation of this single regulatory protein is prevented, oxidant-induced apoptosis is inhibited. Oxidation of cofilin causes it to lose its affinity for actin and to translocate to the mitochondria, where it induces swelling and cytochrome c release by mediating opening of the permeability transition pore (PTP). This occurs independently of Bax activation and requires both oxidation of cofilin Cys residues and dephosphorylation at Ser 3. Knockdown of endogenous cofilin using targeted siRNA inhibits oxidant-induced apoptosis, which is restored by re-expression of wild-type cofilin but not by cofilin containing Cys to Ala mutations. Exposure of cofilin to TnCl results in intramolecular disulphide bonding and oxidation of Met residues to Met sulphoxide, but only Cys oxidation causes cofilin to induce mitochondrial damage.

Rho GDP dissociation inhibitor (RhoGDI) plays an essential role in control of a variety of cellular functions through interactions with Rho family GTPases, including Rac1, Cdc42, and RhoA. RhoGDI is frequently overexpressed in human tumors and chemo-resistant cancer cell lines, raising the possibility that RhoGDI might play a role in the development of drug resistance in cancer cells. We found that overexpression of RhoGDI increased resistance of cancer cells (MDA-MB-231 human breast cancer cells and JLP-119 lymphoma cells) to the induction of apoptosis by two chemotherapeutic agents: etoposide and doxorubicin. Conversely, silencing of RhoGDI expression by DNA vector-mediated RNA interference (small interfering RNA) sensitized MDA-MB-231 cells to drug-induced apoptosis. Resistance to apoptosis was restored by reintroduction of RhoGDI protein expression. The mechanism for the anti-apoptotic activity of RhoGDI may derive from its ability to inhibit caspase-mediated cleavage of Rac1 GTPase, which is required for maximal apoptosis to occur in response to cytotoxic drugs. Taken together, the data show that RhoGDI is an anti-apoptotic molecule that mediates cellular resistance to these chemotherapy agents.

The small GTPase Rac1 has emerged as an important regulator of cell survival and apoptosis, but the mechanisms involved are not completely understood. In this report, constitutively active Rac1 is shown to stimulate the phosphorylation of the Bcl-2 family member Bad, thereby suppressing drug-induced caspase activation and apoptosis in human lymphoma cells. Rac1 activation leads to human Bad phosphorylation specifically at serine-75 (corresponding to murine serine-112) both in vivo and in vitro. Inhibition of constitutive and activated Rac1-induced Bad phosphorylation by a cell-permeable competitive peptide inhibitor representing this Bad phosphorylation site sensitizes lymphoma cells to drug-induced apoptosis. The data show further that endogenous protein kinase A is a primary catalyst of cellular Bad phosphorylation in response to Rac activation, while Akt is not involved. These findings define a mechanism by which active Rac1 promotes lymphoma cell survival and inhibits apoptosis in response to cancer chemotherapy drugs.

The Rac members of the Rho family GTPases control signaling pathways that regulate diverse cellular activities, including cytoskeletal organization, gene transcription, and cell transformation. Rac is implicated in apoptosis, but little is known about the mechanism by which it responds to apoptotic stimuli. Here we demonstrate that endogenous Rac GTPases are caspase 3 substrates that are cleaved in human lymphoma cells during drug-induced apoptosis. Cleavage of Rac1 occurs at two unconventional caspase 3 sites, VVGD11/G and VMVD47/G, and results in inactivation of the GTPase and effector functions of the protein (binding to the p21-activated protein kinase PAK1). Expression of caspase 3-resistant Rac1 mutants in the cells suppresses drug-induced apoptosis. Thus, proteolytic inactivation of Rac GTPases represents a novel, irreversible mechanism of Rac downregulation that allows maximal cell death following drug treatment.

Background: Substantial clinical and preclinical evidence have indicated the association between amide-linked local anesthesia and the long-term outcomes of cancer patients. However, the potential effects of local anesthesia on cancer recurrence are inconclusive and the underlying mechanisms remain poorly understood. Methods: We systematically examined the effects of three commonly used local anesthetics in melanoma cells and analyzed the underlying mechanisms focusing on small GTPases. Results: Ropivacaine and lidocaine but not bupivacaine inhibited migration and proliferation, and induced apoptosis in melanoma cells. In addition, ropivacaine and lidocaine but not bupivacaine significantly augmented the in vitro efficacy of vemurafenib (a B-Raf inhibitor for melanoma with BRAF V600E mutation) and dacarbazine (a chemotherapeutic drug). Mechanistically, ropivacaine but not bupivacaine decreased the activities of Ras superfamily members with the dominant inhibitory effects on RhoA and Ras, independent of sodium channel blockade. Rescue studies using constitutively active Ras and Rho activator calpeptin demonstrated that ropivacaine inhibited migration mainly through RhoA whereas growth and survival were mainly inhibited through Ras in melanoma cells. We further detected a global reduction of downstream signaling of Ras and RhoA in ropivacaine-treated melanoma cells. Conclusion: Our study is the first to demonstrate the anti-melanoma activity of ropivacaine and lidocaine but not bupivacaine, via targeting small GTPases. Our findings provide preclinical evidence on how amide-linked local anesthetics could affect melanoma patients. [ABSTRACT FROM AUTHOR]

To investigate the therapeutic mechanisms underlying prostaglandin E1 (PGE1) and angiotensin-converting enzyme inhibitor (ACEI) on reducing urinary protein in diabetic kidney disease (DKD). DKD rats were established and randomly divided into four groups: PGE1 (10 μg/kg/day) (P group), ACEI (10 mg/kg/day) (A group), combination of PGE1 with ACEI treatment (P + A group), and saline treatment group (DKD group). Untreated rats were used as normal control (N group). Urinary albumin, endothelin-1 (ET-1), angiotensin II (AngII), TUNEL assay, Masson's trichrome staining, and immunohistochemistry staining for CD68 were evaluated in all groups. Ten days after treatment, urinary albumin was significantly decreased in the P and P + A groups ( p < 0.01 vs. the DKD group ). At the end of 8 weeks, the albumin was still significantly reduced in the P + A group ( p < 0.05 vs. the A group). ET-1 and AngII were also significantly decreased in three treatment groups ( p < 0.01 vs. the DKD group), especially in the P + A group. Few cells underwent apoptosis in glomerular regions in DKD rats, while amounts of apoptotic cells were seen in tubules regions. Further, apoptosis and the areas of fibrosis in tubulointerstitial were both decreased most in the P + A group compared with the DKD group. Apoptosis of renal tubular epithelial cells may participate in the development and progression of DKD in rats. Combination of PGE1 with AGEI remarkably protects renal function compared with PGE1 or ACEI monotherapy. The potential therapeutic mechanisms of PGE1 and AGEI might be via multiple targets and, at least in part, through inhibiting the apoptosis of renal tubular epithelial cells. [ABSTRACT FROM AUTHOR]

Pancreatic β-cell death or dysfunction mediated by oxidative stress underlies the development and progression of diabetes mellitus ( DM). In this study, we evaluated the effect of lentinan ( LNT), an active ingredient purified from the bodies of Lentinus edodes, on pancreatic β-cell apoptosis and dysfunction caused by streptozotocin ( STZ) and the possible mechanisms implicated. The rat insulinoma cell line INS-1 were pre-treated with the indicated concentration of LNT for 30 min. and then incubated for 24 hrs with or without 0.5 mM STZ. We found that STZ treatment causes apoptosis of INS-1 cells by enhancement of intracellular reactive oxygen species ( ROS) accumulation, inducible nitric oxide synthase ( iNOS) expression and nitric oxide release and activation of the c-jun N-terminal kinase ( JNK) and p38 mitogen-activated protein kinase ( MAPK) signalling pathways. However, LNT significantly increased cell viability and effectively attenuated STZ-induced ROS production, iNOS expression and nitric oxide release and the activation of JNK and p38 MAPK in a dose-dependent manner in vitro. Moreover, LNT dose-dependently prevented STZ-induced inhibition of insulin synthesis by blocking the activation of nuclear factor kappa beta and increasing the level of Pdx-1 in INS-1 cells. Together these findings suggest that LNT could protect against pancreatic β-cell apoptosis and dysfunction caused by STZ and therefore may be a potential pharmacological agent for preventing pancreatic β-cell damage caused by oxidative stress associated with diabetes. [ABSTRACT FROM AUTHOR]