Anti-tumor activity of CrTX in human lung adenocarcinoma cell line A549

Aim:

To assess the cytotoxic effect of crotoxin (CrTX), a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus, in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms.

Methods:

A549 cells were treated with gradient concentrations of CrTX, and the cell cycle and apoptosis were analyzed using a flow cytometric assay. The changes of cellular effectors p53, caspase-3 and cleaved caspase-3, total P38MAPK and pP38MAPK were investigated using Western blot assays. A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo.

Results:

Treatment of A549 cells with CrTX (25–200 μg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC₅₀=78 μg/mL). Treatment with CrTX (25 μg/mL) for 24 h caused G1 arrest and induced cell apoptosis. CrTX (25 μg/mL) significantly increased the expression of wt p53, cleaved caspase-3 and phospho-P38MAPK. Pretreatment with the specific P38MAPK inhibitor SB203580 (5 μmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level, but G₁ arrest remained unchanged and highly expressed p53 sustained. Intraperitoneal injection of CrTX (10 μg/kg, twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth, and decreased MVD and VEGF levels.

Conclusion:

CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3, and by cell cycle arrest mediated by increased wt p53 expression. In addition, CrTX displayed anti-angiogenic effects in vivo.     

Hyperoside induces both autophagy and apoptosis in non-small cell lung cancer cells in vitro

Aim:

Hyperoside (quercetin-3-O-β-D-galactopyranoside) is a flavonol glycoside found in plants of the genera Hypericum and Crataegus, which exhibits anticancer, anti-oxidant, and anti-inflammatory activities. In this study we investigated whether autophagy was involved in the anticancer mechanisms of hyperoside in human non-small cell lung cancer cells in vitro.

Methods:

Human non-small cell lung cancer cell line A549 was tested, and human bronchial epithelial cell line BEAS-2B was used for comparison. The expression of LC3-II, apoptotic and signaling proteins was measured using Western blotting. Autophagosomes were observed with MDC staining, LC3 immunocytochemistry, and GFP-LC3 fusion protein techniques. Cell viability was assessed using MTT assay.

Results:

Hyperoside (0.5, 1, 2 mmol/L) dose-dependently increased the expression of LC3-II and autophagosome numbers in A549 cells, but had no such effects in BEAS-2B cells. Moreover, hyperoside dose-dependently inhibited the phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, but increased the phosphorylation of ERK1/2 in A549 cells. Insulin (200 nmol/L) markedly enhanced the phosphorylation of Akt and decreased LC3-II expression in A549 cells, which were reversed by pretreatment with hyperoside, whereas the MEK1/2 inhibitor U0126 (20 μmol/L) did not blocked hyperoside-induced LC3-II expression. Finally, hyperoside dose-dependently suppressed the cell viability and induced apoptosis in A549 cells, which were significantly attenuated by pretreatment with the autophagy inhibitor 3-methyladenine (2.5 mmol/L).

Conclusion:

Hyperoside induces both autophagy and apoptosis in human non-small cell lung cancer cells in vitro. The autophagy is induced through inhibiting the Akt/mTOR/p70S6K signal pathways, which contributes to anticancer actions of hyperoside.     

β, β-Dimethylacrylshikonin induces mitochondria-dependent apoptosis of human lung adenocarcinoma cells in vitro via p38 pathway activation

Aim:

β, β-Dimethylacrylshikonin (DMAS) is an anticancer compound extracted from the roots of Lithospermum erythrorhizon. In the present study, we investigated the effects of DMAS on human lung adenocarcinoma cells in vitro and explored the mechanisms of its anti-cancer action.

Methods:

Human lung adenocarcinoma A549 cells were tested. Cell viability was assessed using an MTT assay, and cell apoptosis was evaluated with flow cytometry and DAPI staining. The expression of the related proteins was detected using Western blotting. The mitochondrial membrane potential was measured using a JC-1 kit, and subcellular distribution of cytochrome c was analyzed using immunofluorescence staining.

Results:

Treatment of A549 cells with DMAS suppressed the cell viability in dose- and time-dependent manners (the IC₅₀ value was 14.22 and 10.61 μmol/L, respectively, at 24 and 48 h). DMAS (7.5, 10, and 15 μmol/L) dose-dependently induced apoptosis, down-regulated cIAP-2 and XIAP expression, and up-regulated Bax and Bak expression in the cells. Furthermore, DMAS resulted in loss of mitochondrial membrane potential and release of cytochrome c in the cells, and activated caspase-9, caspase-8, and caspase-3, and subsequently cleaved PARP, which was abolished by pretreatment with Z-VAD-FMK, a pan-caspase inhibitor. DMAS induced sustained p38 phosphorylation in the cells, while pretreatment with SB203580, a specific p38 inhibitor, blocked DMAS-induced p38 activation and apoptosis.

Conclusion:

DMAS inhibits the growth of human lung adenocarcinoma A549 cells in vitro via activation of p38 signaling pathway.     

Fructus phyllanthi tannin fraction induces apoptosis and inhibits migration and invasion of human lung squamous carcinoma cells in vitro via MAPK/MMP pathways

Aim:

Fructus phyllanthi tannin fraction (PTF) from the traditional Tibetan medicine Fructus phyllanthi has been found to inhibit lung and liver carcinoma in mice. In this study we investigated the anticancer mechanisms of PTF in human lung squamous carcinoma cells in vitro.

Methods:

Human lung squamous carcinoma cell line (NCI-H1703), human large-cell lung cancer cell line (NCI-H460), human lung adenocarcinoma cell line (A549) and human fibrosarcoma cell line (HT1080) were tested. Cell viability was detected with MTT assay. Cell migration and invasion were assessed using a wound healing assay and a transwell chemotaxis chambers assay, respectively. Cell apoptosis was analyzed with flow cytometric analysis. The levels of apoptosis-related and metastasis-related proteins were detected by Western blot and immunofluorescence.

Results:

PTF dose-dependently inhibited the viability of the 3 human lung cancer cells. The IC₅₀ values of PTF in inhibition of NCI-H1703, NCI-H460, and A549 cells were 33, 203, and 94 mg/L, respectively. PTF (15, 30, and 60 mg/L) dose-dependently induced apoptosis of NCI-H1703 cells. Treatment of NCI-H1703 and HT1080 cells with PTF significantly inhibited cell migration, and reduced the number of invasive cells through Matrigel. Furthermore, PTF dose-dependently down-regulated the expression of phosphor-ERK1/2, MMP-2 and MMP-9, up-regulated the expression of phosphor-JNK, but had no significant effect on the expression of ERK1/2 or JNK.

Conclusion:

PTF induces cell apoptosis and inhibits the migration and invasion of NCI-H1703 cells by decreasing MPPs expression through regulation of the MAPK pathway.     

Down-regulation of ALKBH2 increases cisplatin sensitivity in H1299 lung cancer cells

Aim:

To elucidate the combined effect of alkylated DNA repair protein alkB homolog 2 (ALKBH2)-targeting gene therapy and cisplatin (cDDP) chemotherapy on the non-small cell lung cancer (NSCLC) H1299 cell line.

Methods:

ALKBH2 was down-regulated in H1299 cells by lentivirus-mediated RNA interference (RNAi). Changes in ALKBH2 expression were determined using real-time RT-PCR and Western blotting. Cell viability was evaluated using MTT assay. DNA synthesis in proliferating cells was determined using BrdU incorporation assay. Cell apoptosis was determined using flow cytometry.

Results:

Lentivirus-mediated ALKBH2 silencing alone did not induce apoptosis or attenuate the growth potential of H1299 cells within five days post-infection. Combined treatment modalities with lentivirus-mediated ALKBH2 down-regulation and cDDP (333 μmol/L) were significantly more potent in inhibiting cell growth and inducing apoptosis than mono-chemotherapy.

Conclusion:

Combined treatment modalities of ALKBH2 knockdown and cDDP chemotherapy have the potential to improve the efficacy in the treatment of NSCLC.     

Genistein enhances the effect of trichostatin A on inhibition of A549 cell growth by increasing expression of TNF receptor-1

Our previous study has shown that genistein enhances apoptosis in A549 lung cancer cells induced by trichostatin A (TSA). The precise molecular mechanism underlying the effect of genistein, however, remains unclear. In the present study, we investigated whether genistein enhances the anti-cancer effect of TSA through up-regulation of TNF receptor-1 (TNFR-1) death receptor signaling. We incubated A549 cells with TSA (50 ng/mL) alone or in combination with genistein and then determined the mRNA and protein expression of TNFR-1 as well as the activation of downstream caspases. Genistein at 5 and 10 μM significantly enhanced the TSA-induced decrease in cell number and apoptosis in a dose-dependent manner. The combined treatment significantly increased mRNA and protein expression of TNFR-1 at 6 and 12 h, respectively, compared with that of the control group; while TSA alone had no effect. TSA in combination with 10 μM of genistein increased TNFR-1 mRNA and protein expression by about 70% and 40%, respectively. The underlying mechanism for this effect of genistein may be partly associated with the estrogen receptor pathway. The combined treatment also increased the activation of caspase-3 and ‐10 as well as p53 protein expression in A549 cells. The enhancing effects of genistein on the TSA-induced decrease in cell number and on the expression of caspase-3 in A549 cells were suppressed by silencing TNFR-1 expression. These data demonstrated that the upregulation of TNFR-1 death receptor signaling plays an important role, at least in part, in the enhancing effect of genistein on TSA-induced apoptosis in A549 cells.     

Migfilin sensitizes cisplatin-induced apoptosis in human glioma cells in vitro

Aim:

Filamin binding LIM protein 1, also known as migfilin, is a skeleton organization protein that binds to mitogen-inducible gene 2 at cell-extracellular matrix adhesions. The aim of this study was to investigate the role of migfilin in cisplatin-induced apoptosis in human glioma cells, to determine the functional domains of migfilin, and to elucidate the molecular mechanisms underlying the regulation of cisplatin-related chemosensitivity.

Methods:

The human glioma cell lines Hs683, H4, and U-87 MG were transfected with pEGFP-C2-migfilin to elevate the expression level of migfilin. RNA interference was used to reduce the expression of migfilin. To determine the functional domains of migfilin, U-87 MG cells were transfected with plasmids of migfilin deletion mutants. After treatment with cisplatin (40 μmol/L) for 24 h, the cell viability was assessed using the MTS assay, and the cell apoptotic was examined using the DAPI staining assay and TUNEL analysis. Expression levels of apoptosis-related proteins were detected by Western blot analysis.

Results:

Overexpression of migfilin significantly enhanced cisplatin-induced apoptosis in Hs683, H4, and U-87 MG cells, whereas downregulation of migfilin expression inhibited the chemosensitivity of these cell lines. The N-terminal region of migfilin alone was able to enhance the cisplatin-induced apoptosis. However, despite the existence of the N-terminal region, mutants of migfilin with any one of three LIM domains deleted led to a function loss. Furthermore, apoptotic proteins (PARP and caspase-3) and the anti-apoptotic protein Bcl-xL were modulated by the expression level of migfilin in combination with cisplatin.

Conclusion:

The LIM1-3 domains of migfilin play a key role in sensitizing glioma cells to cisplatin-induced apoptosis through regulation of apoptosis-related proteins.     

Resveratrol analogue 3,4,4′-trihydroxy-trans-stilbene induces apoptosis and autophagy in human non-small-cell lung cancer cells in vitro

Aim:

To investigate the effects of 3,4,4′-trihydroxy-trans-stilbene (3,4,4′-THS), an analogue of resveratrol, on human non-small-cell lung cancer (NSCLC) cells in vitro.

Methods:

Cell viability of NSCLC A549 cells was determined by MTT assay. Cell apoptosis was evaluated using flow cytometry and TUNEL assay. Cell necrosis was evaluated with LDH assay. The expression of apoptosis- or autophagy-associated proteins was measured using Western blotting. The formation of acidic compartments was detected using AO staining, neutral red staining and Lysotracker-Red staining. LC3 punctae were analyzed with fluorescence microscopy.

Results:

Treatment with 3,4,4′-THS (10-80 μmol/L) concentration-dependently inhibited the cell viability. It did not cause cell necrosis, but induced apoptosis accompanied by up-regulation of cleavaged PARP, caspase3/9 and Bax, and by down-regulation of Bcl-2 and surviving. It also increased the formation of acidic compartments, LC3-II accumulation and GFP-LC3 labeled autophagosomes in the cells. It inhibited the mTOR-dependent pathway, but did not impair autophagic flux. 3,4,4′-THS-induced cell death was enhanced by the autophagy inhibitors 3-MA (5 mmol/L) or Wortmannin (2 μmol/L). Moreover, 3,4,4′-THS treatment elevated the ROS levels in the cells, and co-treatment with 3-MA further elevated the ROS levels. 3,4,4′-THS-induced apoptosis and autophagy in the cells was attenuated by NAC (10 mmol/L)

Conclusion:

3,4,4′-THS induces both apoptosis and autophagy in NSCLC A549 cells in vitro. Autophagy inhibitors promote 3,4,4′-THS-induced apoptosis of A549 cells, thus combination of 3,4,4′-THS and autophagy inhibitor provides a promising strategy for NSCLC treatment.     

Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor-1α expression through the induction of glucocorticoidinduced leucine zipper

Background and Purpose

The COX-2/PGE2 pathway in hypoxic cancer cells has important implications for stimulation of inflammation and tumourigenesis. However, the mechanism by which glucocorticoid receptors (GRs) inhibit COX-2 during hypoxia has not been elucidated. Hence, we explored the mechanisms underlying glucocorticoid-mediated inhibition of hypoxia-induced COX-2 in human distal lung epithelial A549 cells.

Experimental Approach

The expressions of COX-2 and glucocorticoid-induced leucine zipper (GILZ) in A549 cells were determined by Western blot and/or quantitative real time-PCR respectively. The anti-invasive effect of GILZ on A549 cells was evaluated using the matrigel invasion assay.

Key Results

The hypoxia-induced increase in COX-2 protein and mRNA levels and promoter activity were suppressed by dexamethasone, and this effect of dexamethasone was antagonized by the GR antagonist RU486. Overexpression of GILZ in A549 cells also inhibited hypoxia-induced COX-2 expression levels and knockdown of GILZ reduced the glucocorticoid-mediated inhibition of hypoxia-induced COX-2 expression, indicating that the inhibitory effects of dexamethasone on hypoxia-induced COX-2 are mediated by GILZ. GILZ suppressed the expression of hypoxia inducible factor (HIF)-1α at the protein level and affected its signalling pathway. Hypoxia-induced cell invasion was also dramatically reduced by GILZ expression.

Conclusion and Implications

Dexamethasone-induced upregulation of GILZ not only inhibits the hypoxic-evoked induction of COX-2 expression and cell invasion but further blocks the HIF-1 pathway by destabilizing HIF-1α expression. Taken together, these findings suggest that the suppression of hypoxia-induced COX-2 by glucocorticoids is mediated by GILZ. Hence, GILZ is a potential key therapeutic target for suppression of inflammation under hypoxia.     

Characterization of the anticancer effects of S115, a novel heteroaromatic thiosemicarbazone compound,in vitro and in vivo

Aim:

To investigate the anticancer effects of S115, a novel heteroaromatic thiosemicarbazone compound in vitro and in vivo.

Methods:

The anti-proliferative action of S115 was analyzed in 12 human and mouse cancer cell lines using MTT assay. Autograft and xenograft cancer models were made by subcutaneous inoculation of cancer cells into mice or nude mice. The mice were orally treated with S115 (2, 8, 32 mg·kg⁻¹·d⁻¹) for 7 d, and the tumor size was measured every 3 d. Cell apoptosis and cell cycle distribution were examined using flow cytometry, gene expression profile analyses, Western blots and RT-PCR.

Results:

The IC₅₀ values of S115 against 12 human and mouse cancer cell lines ranged from 0.3 to 6.6 μmol/L. The tumor growth inhibition rate caused by oral administration of S115 (32 mg·kg⁻¹·d⁻¹) were 89.7%, 81.7%, 78.4% and 77.8%, respectively, in mouse model of B16 melanoma, mouse model of Colon26 colon cancer, nude mouse model of A549 lung cancer and nude mouse model of SK-OV-3 ovarian cancer. Furthermore, oral administration of S115 (7.5 mg·kg⁻¹·d⁻¹) synergistically enhanced the anticancer effects of cyclophosphamide, cisplatin, or 5-fluorouracil in mouse model of S180 sarcoma. Treatment of A549 human lung cancer cells with S115 (1.5 μmol/L) induced G₀/G₁ cell cycle arrest, and increased apoptosis. Furthermore, S115 downregulated the level of ubiquitin, and upregulated the level of Tob2 in A549 cells.

Conclusion:

S115 exerts anticancer effects against a variety of cancer cells in vitro and in grafted cancer models by inducing apoptosis, downregulating ubiquitin and upregulating Tob2.     

Huperzine A derivative M3 protects PC12 cells against sodium nitroprusside-induced apoptosis

Aim:

To investigate the effects of M3, a derivative of huperzine A, on the apoptosis induced by sodium nitroprusside (SNP) in PC12 cells.

Methods:

Cell viability was detected using MTT method. Apoptosis was examined with annexin V/prodium iodide (PI) stain. The levels of reactive oxygen species (ROS) were measured using fluorophotometric quantitation. The amount of malonaldehyde (MDA) was determined with MDA detection kits. The expression of caspase-3 and Hsp70 were analyzed using Western blotting.

Results:

Exposure of PC12 cells to SNP (200 μmol/L) for 24 h decreased the cell viability to 69.0% of that in the control group. Pretreatment with M3 (10 μmol/L) or huperzine A (10 μmol/L) significantly protected the cells against SNP-induced injury and apoptosis; the ratio of apoptotic bodies in PC12 cells was decreased from 27.3% to 15.0%. Pretreatment with M3 (10 μmol/L) significantly decreased ROS and MDA levels, and increased the expression of Hsp70 in the cells. Quercetin (10 μmol/L) blocked the protective effect of M3, while did not influence on that of huperzine A.

Conclusion:

M3 protects PC12 cells against SNP-induced apoptosis, possible due to ROS scavenging and Hsp70 induction.     

A novel quinone-based derivative (DTNQ-Pro) induces apoptotic death via modulation of heat shock protein expression in Caco-2 cells

Background and purpose:

The resistance of human colon adenocarcinoma cells to antineoplastic agents may be related to the high endogenous expression of stress proteins, including the family of heat shock proteins (HSPs). Recently, a quinone-based pentacyclic derivative, DTNQ-Pro, showed high cytotoxic activity in human colon carcinoma cell lines. The aim of the present study was to determine the precise cellular mechanisms of this cytotoxic action of DTNQ-Pro.

Experimental approach:

Using human colorectal carcinoma-derived Caco-2 cells as a model, we studied the effects of DTNQ-Pro on cellular viability and oxidative stress; HSP70 and HSP27 accumulation; and cell cycle, differentiation and apoptosis.

Key results:

Incubation of Caco-2 cells with DTNQ-Pro reduced cell growth and increased the levels of reactive oxygen species in mitochondria. After 48 h of treatment, cells surviving showed an increased expression of Mn-superoxide dismutase (SOD), nitric oxide production and membrane lipid peroxidation. Treatment with DTNQ-Pro decreased HSP70 expression, and redistributed HSP27 and vimentin within the cell. DTNQ-Pro down-regulated the expression of A and B cyclins with arrest of the cell cycle in S phase and increased cellular differentiation. A second treatment of Caco-2 cells with DTNQ-Pro induced cellular death by activation of the apoptotic pathway.

Conclusions and implications:

DTNQ-Pro causes Caco-2 cell death by induction of apoptosis via inhibition of HSP70 accumulation and the intracellular redistribution of HSP27. These findings suggest the potential use of DTNQ-Pro in combination chemotherapy for colon cancer.     

Umbelliprenin is cytotoxic against QU-DB large cell lung cancer cell line but anti-proliferative against A549 adenocarcinoma cells

Background

Umbelliprenin is a natural compound, belonging to the class of sesquiterpene coumarins. Recently, umbelliprenin has attracted the researchers'' attention for its antitumor activities against skin tumors. Its effect on lung cancer is largely unknown. The aim of our study was to investigate the effects of this natural compound, which is expected to have low adverse effects, on lung cancer.

Methods

The QU-DB large cell and A549 adenocarcinoma lung cancer cell lines were treated with umbelliprenin. IC₅₀ values were estimated using methyl thiazolely diphenyl-tetrazolium bromide (MTT) assay, in which a decrease in MTT reduction can occur as a result of cell death or cell proliferation inhibition. To quantify the rate of cell death at IC₅₀ values, flow cytometry using Annexin V-FITC (for apoptotic cells), and propidium iodide (for necrotic cells) dyes were employed.

Results

Data from three independent MTT experiments in triplicate revealed that IC₅₀ values for QU-DB and A549 were 47 ± 5.3 μM and 52 ± 1.97 μM, respectively. Annexin V/PI staining demonstrated that umbelliprenin treatment at IC₅₀ induced 50% cell death in QU-DB cells, but produced no significant death in A549 cells until increasing the umbelliprenin concentration to IC₈₀. The pattern of cell death was predominantly apoptosis in both cell lines. When peripheral blood mononuclear cells were treated with 50 μM and less concentrations of umbelliprenin, no suppressive effect was observed.

Conclusions

We found cytotoxic/anti-proliferative effects of umbelliprenin against two different types of lung cancer cell lines.     

S-allylcysteine,a garlic derivative,suppresses proliferation and induces apoptosis in human ovarian cancer cells in vitro

Aim： To investigate the effects of S-allylcysteine （SAC）, a water-soluble garlic derivative, on human ovarian cancer cells in vitro. Methods： Human epithelial ovarian cancer cell line A2780 was tested. Cell proliferation was examined with CCK-8 and colony formation assays. Cell cycle was analyzed with flow cytometry. Cell apoptosis was studied using Hoeohst 33258 staining and Annexin V/PI staining with flow cytometry. The migration and invasion of A2780 cells were examined with transwell and wound healing assays. The expression of relevant proteins was detected with Western blot assays. Results： SAC （1-100 mmol/L） inhibited the proliferation of A2780 cells in dose- and time-dependent manners （the ICsovalue was approximately 25 mmoVL at 48 h, and less than 6.25 mmol/L at 96 h）. Furthermore, SAC dose-dependently inhibited the colony formation of A2780 cells. Treatment of A2780 cells with SAC resulted in G/S phase arrest and induced apoptosis, accompanied by decreased expression of pro-caspase-3, Parp-1 and Bcl-2, and increased expression of active caspase-3 and Bax. SAC treatment significantly reduced the migration of A2780 cells, and markedly decreased the protein expression of Wnt5a, p-AKT and c-Jun, which were the key proteins involved in proliferation and metastasis. Conclusion： SAC suppresses proliferation and induces apoptosis in A2780 ovarian cancer cells in vitro.     

Mig-2 attenuates cisplatin-induced apoptosis of human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways

Aim:

Mig-2 (also known as Kindlin-2 and FERMT2) is an important regulator of integrin activation and cell-extracellular matrix adhesion, and involved in carcinogenesis and tumor progression. The aim of this study was to investigate the role of mig-2 in cisplatin-induced apoptosis of human glioma cells in vitro.

Methods:

The expression of mig-2 was modulated in human glioma H4, HS 683 and U-87 MG cells by transfection with a plasmid carrying mig-2 or mig-2 siRNA. Cisplatin-induced apoptosis was detected using Annexin V/PI staining and flow cytometry, as well as MTS analyses. The expression of apoptosis-related or signaling proteins was examined using Western blotting analysis. H4 cells were transfected with plasmids carrying mig-2 mutants to determine the functional domain of mig-2.

Results:

In the 3 glioma cell lines tested, overexpression of mig-2 significantly attenuated cisplatin-induced apoptosis, whereas knock-down of mig-2 potentiated the apoptosis. The mechanisms of action of mig-2 were further addressed in H4 cells: overexpression of mig-2 markedly reduced cleaved caspase-9, caspase-8, caspase-3 and PARP, as well as p-JNK and p-p38, and increased p-AKT in cisplatin-treated H4 cells, whereas mig-2 siRNA reversely changed these apoptosis-related and signaling proteins. Furthermore, pretreatment with JNK inhibitor SP600125 and p38 inhibitor SB203580, or with AKT inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 abolished the effects of mig-2 on cisplaxtin-induced apoptosis. In H4 cells, GFP-mig-2 F3 plasmid that contained only the F3 subdomain showed the same efficiency in attenuating cisplatin-induced apoptosis, as the mig-2 wild-type vector did, whereas GFP-mig-2 (1-541) plasmid that lacked the F3 subdomain was inactive.

Conclusion:

Mig-2 significantly attenuates the antitumor action of cisplatin against human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways. The F3 subdomain of mig-2 is necessary and sufficient for this effect.     

CYP3A4 overexpression enhances the cytotoxicity of the antitumor triazoloacridinone derivative C-1305 in CHO cells

Aim:

To examine how the higher expression level of CYP3A4 isoenzyme influenced the cytotoxicity of the antitumor triazoloacridinone derivative C-1305 in Chinese hamster ovary (CHO) cells.

Methods:

Three CHO cell lines were examined: wild-type CHO cells; CHO-HR cells with overexpression of human cytochrome P450 reductase (CPR); and CHO-HR-3A4 cells with coexpression of human CYP3A4 and CPR. Cellular responses caused by C-1305 were monitored using DAPI staining, cell cycle analysis, phosphatydilserine externalization analysis and SA-β-galactosidase expression analysis. Cell viability was assessed with simultaneous FDA and PI staining.

Results:

Treatment with C-1305 for 72 h exhibited different levels of cytotoxicity in the 3 cell lines, and the values of IC80 in CHO, CHO-HR and CHO-HR-3A4 cells were 0.087±0.005, 0.032±0.0001, and 0.064±0.0095 μmol/L, respectively. The cell cycle analysis revealed that both CHO and CHO-HR cells underwent transient G₂/M arrest, whereas CHO-HR-3A4 cells did not accumulate in this phase. Prolonged exposure up to 120 h caused time-dependent increase in the sub-G₁ fraction in all the 3 cell lines. Treatment with C-1305 caused cell death through apoptosis and necrosis. However, these processes were more pronounced in the transfected CHO cells than in the wild-type cells. The cells surviving after C-1305 exposure underwent senescence.

Conclusion:

CYP3A4 overexpression potently enhances the cellular responses (apoptosis, necrosis and senescence) caused by C-1305 in CHO cells.     

Differential transport of platinum compounds by the human organic cation transporter hOCT2 (hSLC22A2)

Background:

Solute carriers (SLCs), in particular organic cation transporters (OCTs), have been implicated in the cellular uptake of platinum-containing anticancer compounds. The activity of these carriers may determine the pharmacokinetics and the severity of side effects, including neuro- and nephrotoxicity of platinum-based chemotherapy. As decreased drug accumulation is a key mechanism of platinum resistance, SLCs may also contribute to the development of resistance. Here, we define the role of hSLC22A2 (OCT2) in the cellular uptake of platinum compounds.

Experimental approach:

Human embryonic kidney (HEK) 293 cells stably expressing the hSLC22A2 gene (HEK293/hSLC22A2) were used in platinum accumulation studies. Following a 2 h exposure to various platinum compounds (100 µM), intracellular platinum levels were determined by flameless atomic absorption spectrometry.

Key results:

HEK293/hSLC22A2 cells, compared with HEK293/Neo control cells, displayed significant increases in oxaliplatin (28.6-fold), Pt[DACH]Cl₂ (20.6-fold), ormaplatin (8.1-fold), tetraplatin (4.5-fold), transplatin (3.7-fold) and cisplatin (1.3-fold), but not carboplatin. SLC22A2-mediated transport could be inhibited by 1-methyl-4-phenylpyridinium. Furthermore, hSLC22A2-mediated oxaliplatin and cisplatin accumulation was time- and concentration-dependent, but non-saturable. Expression of hSLC22A2 in HEK293 cells resulted in enhanced sensitivity to oxaliplatin (12-fold) and cisplatin (1.8-fold). Although, hSLC22A2 mRNA expression was frequently found in ovarian cancer cell lines, its expression in clinical ovarian cancer specimens (n= 80) was low and did not correlate with the treatment outcome of platinum-based regimens.

Conclusions and implications:

The hSLC22A2 drug transporter is a critical determinant in the uptake and cytotoxicity of various platinum compounds, particularly oxaliplatin.