Meiling Yu

Cx43 reverses the resistance of A549 lung adenocarcinoma cells to cisplatin by inhibiting EMT.

Cisplatin (CDDP) is one of the standard first-line chemotherapeutic agents for advanced non-small cell lung cancer (NSCLC). Unfortunately, prolonged exposure to CDDP results in acquired resistance which prevents the successful treatment of lung cancer patients. Thus, it is necessary to explore the mechanism underlying the resistance of NSCLC to CDDP. In the present study, a CDDP-resistant human lung cancer cell line A549/CDDP was established from the parental cell line A549. The results demonstrated that A549/CDDP cells acquired an epithelial-mesenchymal transition (EMT) phenotype, with morphological changes including acquisition of a spindle-like fibroblastic phenotype, downregulation of E-cadherin, upregulation of mesenchymal markers (vimentin, Snail and Slug), and increased capability of invasion and migration. Compared with A549 cells, the A549/CDDP cells showed decreased connexin43 (Cx43) expression. Overexpression of Cx43 reversed EMT and CDDP resistance in the A549/CDDP cells. Conversely, knockdown of Cx43 expression by siRNA-Cx43 initiated EMT and induced CDDP insensitivity in A549 cells. In summary, Cx43 reverses CDDP resistance in A549 CDDP-resistant cells by preventing EMT, making Cx43 a possible therapeutic target for lung cancer.

Suppression of PMA-induced tumor cell invasion and migration by ginsenoside Rg1 via the inhibition of NF-κB-dependent MMP-9 expression.

Ginseng has become one of the most commonly used alternative herbal medicines, and its active component, ginsenoside Rg1 has known pharmacological effects, including anticancer properties. However, the effects of ginsenoside Rg1 on metastasis have yet to be investigated. In this study, we demonstrated the ability of ginsenoside Rg1 to suppress phorbol myristate acetate (PMA)-induced invasion and migration in MCF-7 breast cancer cells. MCF-7 cells were treated with ginsenoside Rg1 and incubated with or without PMA. The protein and mRNA expression of MMP-9 and MMP-2 was analyzed using Transwell and wound-healing assays and western blotting. The results showed that suppression was associated with the reduced secretion of MMP-9, a key metastatic enzyme. MMP-9 levels were regulated transcriptionally and correlated with the suppression of NF-κB phosphorylation and DNA binding activity. Conversely, ginsenoside Rg1 did not affect MMP-2 mRNA and TIMP-1 mRNA levels, or the activation of AP-1, suggesting a specificity of pathway inhibition. Inhibition of NF-κB activation by p65 small-interfering RNA (siRNA) was shown to suppress PMA-induced cell invasion and migration. The siRNA studies also showed that PMA-induced MMP-9 expression is NF-κB-dependent. The results suggested that the anticancer properties of ginsenoside Rg1 may derive from its ability to inhibit invasion and migration, and that these processes are regulated in breast cancer cells through the NF-κB-mediated regulation of MMP-9 expression.

Role of heteromeric gap junctions in the cytotoxicity of cisplatin.

In several systems, the presence of gap junctions made of a single connexin has been shown to enhance the cytotoxicity of cisplatin. However, most gap junction channels in vivo appear to be heteromeric (composed of more than one connexin isoform). Here we explore in HeLa cells the cytotoxicity to cisplatin that is enhanced by heteromeric gap junctions composed of Cx26 and Cx32, which have been shown to be more selective among biological permeants than the corresponding homomeric channels. We found that survival and subsequent proliferation of cells exposed to cisplatin were substantially reduced when gap junctions were present than when there were no gap junctions. Functional inhibition of gap junctions by oleamide enhanced survival/proliferation, and enhancement of gap junctions by retinoic acid decreased survival/proliferation. These effects occurred only in high density cultures, and the treatments were without effect when there was no opportunity for gap junction formation. The presence of functional gap junctions enhanced apoptosis as reflected in markers of both early-stage and late-stage apoptosis. Furthermore, analysis of caspases 3, 8 and 9 showed that functional gap junctions specifically induced apoptosis by the mitochondrial pathway. These results demonstrate that heteromeric Cx26/Cx32 gap junctions increase the cytotoxicity of cisplatin by induction of apoptosis via the mitochondrial pathway.

Simvastatin-induced up-regulation of gap junctions composed of connexin 43 sensitize Leydig tumor cells to etoposide: An involvement of PKC pathway

Some of lipophilic statins have been reported to enhance toxicities induced by antineoplastic agents but the underling mechanism is unclear. The authors investigated the involvement of Cx43-mediated gap junction intercellular communication (GJIC) in the effect of simvastatin on the cellular toxicity induced by etoposide in this study. The results showed that a major component of the cytotoxicity of therapeutic levels of etoposide is mediated by gap junctions composed of connexin 43(Cx43) and simvastatin at the dosage which does not induce cytotoxicity enhances etoposide toxicity by increasing gap junction coupling. The augmentative effect of simvastatin on GJIC was related to the inhibition of PKC-mediated Cx43 phosphorylation at ser368 and subsequent enhancement of Cx43 membrane location induced by the agent. The present study suggests the possibility that upregulation of gap junctions may be utilized to increase the efficacy of anticancer chemotherapies.

Role of gap junction intercellular communication in testicular leydig cell apoptosis induced by oxaliplatin via the mitochondrial pathway

Platinum agents are widely used in the chemotherapy of testicular cancer. However, adverse reactions and resistance to such agents have limited their application in antineoplastic treatment. The aim of the present study was to determine the role of gap junction intercellular communication (GJIC) composed of Cx43 on oxaliplatin‑induced survival/apoptosis in mouse leydig normal and cancer cells using MTT, Annexin V/PI double staining assays and western blot analysis. The results showed that GJIC exerted opposite effects on the mouse leydig cancer (I-10) and normal (TM3) cell apoptosis induced by oxaliplatin. In leydig cancer cells, survival of cells exposed to oxaliplatin was substantially reduced when gap junctions formed as compared to no gap junctions. Pharmacological inhibition of gap junctions by oleamide and 18-α-glycyrrhetinic acid resulted in enhanced survival/decreased apoptosis while enhancement of gap junctions by retinoic acid led to decreased survival/increased apoptosis. These effects occurred only in high‑density cultures (gap junction formed), while the pharmacological modulations had no effects when there was no opportunity for gap junction formation. Notably, GJIC played an opposite (protective) role in normal leydig cells survival/apoptosis following exposure to oxaliplatin. Furthermore, this converse oxaliplatin‑inducing apoptosis exerted through the functional gap junction was correlated with the mitochondrial pathway‑related protein Bcl-2/Bax and caspase‑3/9. These results suggested that in testicular leydig normal/cancer cells, GJIC plays an opposite role in oxaliplatin‑induced apoptosis via the mitochondrial pathway.

Ginsenoside Rg1 attenuates invasion and migration by inhibiting transforming growth factor-β1-induced epithelial to mesenchymal transition in HepG2 cells

Ginseng has become one of the most commonly used alternative herbal medicines and its active component, ginsenoside Rg1, possesses known pharmacological effects, including anticancer properties. However, the effects of ginsenoside Rg1 on metastasis have not been investigated. The present study demonstrated that ginsenoside Rg1 was able to suppress transforming growth factor‑β1 (TGF‑β1)‑induced invasion and migration in HepG2 liver cancer cells. This suppression was associated with the inhibition of TGF‑β1‑induced epithelial to mesenchymal transition (EMT). Furthermore, TGF‑β1 induced HepG2 cells to undergo EMT and significantly promoted cell invasion and migration. When cells were pretreated with ginsenoside Rg1 for 24 h and subsequently exposed to TGF‑β1 for 24 h, the results demonstrated that ginsenoside Rg1 inhibited the initiation of TGF‑β1‑induced EMT. In addition, HepG2 cells exhibited a mesenchymal phenotype when exposed to TGF‑β1, but when exposed to ginsenoside Rg1 this effect was reversed and the cells exhibited a classical epithelial morphology. Ginsenoside Rg1 also increased the expression of the epithelial phenotype marker E‑cadherin and repressed the expression of the mesenchymal phenotype marker vimentin. In conclusion, the results of the present study suggest that ginsenoside Rg1 may suppress liver cancer invasion and migration in vitro through inhibiting TGF‑β1‑induced EMT.

Baicalein increases cisplatin sensitivity of A549 lung adenocarcinoma cells via PI3K/Akt/NF-κB pathway

Baicalein, a bioactive flavonoid, exhibits anti-inflammatory and anti-cancer activities. However, few studies reported the interaction of baicalein with chemotherapeutic agents. Our study showed that baicalein significantly enhanced the chemosensitivity of cisplatin (CDDP) in vivo and in vitro. We found that A549/CDDP (resistant to CDDP) cells not only acquired epithelial-mesenchymal transition (EMT) phenotype, but also showed increased NF-κB activity compared with A549 cells (sensitive to CDDP). Our study further demonstrated that PI3K/Akt/NF-κB pathway controlled CDDP resistance via EMT and NF-κB-mediated apoptosis. Baicalein significantly suppressed the PI3K/Akt/NF-κB pathway, leading to conversion of EMT to mesenchymal-epithelial transition (MET, the reciprocal mesenchymal to epithelial transition), and inhibition of NF-κB-mediated antiapoptotic proteins in A549/CDDP cells. In conclusion, our study demonstrated that baicalein reversed the resistance of human A549 lung adenocarcinoma cells to cisplatin by inhibiting EMT and attenuating apoptosis via PI3K/Akt/NF-κB pathway.

Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication

Cisplatin, an important chemotherapeutic agent against testicular germ cell cancer, induces testicular toxicity on Leydig and Sertoli cells, leading to serious side-effects such as azoospermia and infertility. In a previous study, it was found that simvastatin enhanced the sensitivity of Leydig tumor cells to chemotherapeutic toxicity through the enhancement of gap junction functions. In the present study, the effect of simvastatin on the sensitivity of normal Sertoli cells to cisplatin and the role of gap junctions in such effects was investigated. The results showed that, simvastatin attenuated cisplatin toxicity only when cells exhibited high-density culture where gap junctional formation was possible. When gap junction function was decreased by the gap junction inhibitor or by siRNA targeting connexin 43, the protective effect of simvastatin to cisplatin toxicity was substantially attenuated. Simvastatin also enhanced gap junction functions between Sertoli cells. This effect was mediated by the reduction of PKC-mediated connexin phosphorylation, thereby increasing connexin 43 membrane localization. Thus, simvastatin-induced enhancement of gap junction‑mediated intercellular communication attenuated cisplatin toxicity on Sertoli cells. This result indicated that enhancement of gap junction function by simvastatin may have bilateral beneficial effects on cisplatin‑based chemotherapy, enhancing cisplatin killing on cancer while ameliorating the reproduction toxicity.

Components of Panax notoginseng saponins enhance the cytotoxicity of cisplatin via their effects on gap junctions

Previously, Panax notoginseng saponin (PNS)-induced enhancement of gap junction (GJ) formation or function was observed to be responsible for the increased cytotoxic action of cisplatin. PNS has three constituents, ginsenoside Rg1 and Rb1, and notoginsenoside R1. The active compounds in PNS responsible for enhancing the cytotoxicity of cisplatin remain unknown. Thus, the effects of the main components of PNS on the cytotoxicity of cisplatin were investigated, as well as the correlation with the modulation of GJ function in transfected HeLa cells. The cytotoxicity of cisplatin (0.25-1 µg/ml) was increased in the presence of GJs. By contrast, the cytotoxicity of cisplatin was decreased when GJs were inhibited by a GJ blocker or by the inhibition of connexin expression. Ginsenoside Rg1 (100 µM) and notoginsenoside R1 (100 µM) were observed to significantly enhance cisplatin cytotoxicity in cells with functional GJs. Ginsenoside Rb1 had no effect on the cytotoxicity of cisplatin in the presence or absence of functional GJs. Cell exposure to ginsenoside Rg1 and notoginsenoside R1 for 4 h led to significant enhancement of a dye-coupled GJ in a dose-dependent manner; however, no effect was observed in cells exposed to ginsenoside Rb1. The present results indicate that ginsenoside Rg1 and notoginsenoside R1 are the active compounds responsible for enhancing the cytotoxic action of cisplatin induced by PNS in the presence of functional GJs.

In vitro inhibited effect of gap junction composed of Cx43 in the invasion and metastasis of testicular cancer resistanced to cisplatin

The effect of gap junction intercellular communication composed of connexin on cancer invasion/metastasis has been thoroughly explored; however, its effect on testicular cancer resistanced to chemotherapy is still unclear. In this study, we found that the capability of invasion and migration of I-10/DDP (cisplatin (DDP)-resistance) cells were elevated. Furthermore, the expression of Cx43 and the function of gap junction (GJ) in I-10/DDP cells were decreased compared with parental I-10 cells. Pharmacological inhibition of GJs by oleamide (Olea) enhanced invasion and migration. However, enhancement of GJs by retinoic acid (RA) decreased invasion and migration of I-10/DDP cells. To further clarify the invasion/migration inhibited effect of GJ in the testicular cancer resistanced to DDP, GJ function was modulated by overexpression and knockdown of Cx43 expression. Overexpression of Cx43 reduced invasion and migration of I-10/DDP cells. Conversely, knockdown of Cx43 expression increased invasion and migration of I-10/DDP cells. In summary, GJ composed of Cx43 inhibits I-10/DDP cells invasion and migration, and it may become the potential therapeutic target for testicular cancer chemotherapy.