Effects of allyl sulfur compounds and garlic extract on the expression of Bcl-2, Bax, and p53 in non small cell lung cancer cell lines

Allyl sulfur compounds play a major role in the chemoprevention against carcinogenesis. The present study compared the antiproliferative effects of diallyl sulfide (DAS), diallyl disulfide (DADS) and garlic extract on p53-wild type H460 and p53-null type H1299 non small cell lung cancer cells (NSCLC). The DAS and DADS treatment of both H460 and H1299 cells resulted in the highest numbers of cells in apoptotic state as measured by acridine orange staining, however, garlic extract treatment did not induce any significant apoptotic cells by MTT assay. DADS was found to be more effective in inducing apoptosis on NSCLC. The level of p53 protein in H460 cell was increased following DADS treatment. DAS and garlic extract treatment of H460 cells induced a rise in the level of Bax and a fall of Bcl-2 level. These results demonstrate that DAS, DADS and garlic extract are effective in reduction of anti-proliferative gene in NSCLC and suggest that modulation of apoptosis-associated cellular proteins by DAS, DADS and garlic extract may be the mechanism for apoptosis which merit further investigation as potential chemoprevention agents.     

Valproic Acid Promotes Apoptosis and Cisplatin Sensitivity Through Downregulation of H19 Noncoding RNA in Ovarian A2780 Cells

Cisplatin resistance is one of the main limitations in the treatment of ovarian cancer, which is partly mediated by long noncoding RNAs (lncRNAs). H19 is a lncRNA involving in cisplatin resistance in cancers. Valproic acid (VPA) is a commonly used drug for clinical treatment of seizure disorders. In addition, this drug may display its effects through regulation of noncoding RNAs controlling gene expression. The aim of the present study was the investigation of VPA treatment effect on H19 expression in ovarian cancer cells and also the relation of the H19 levels with apoptosis and cisplatin resistance. Briefly, treatment with VPA not only led to significant increase in apoptosis rate, but also increased the cisplatin sensitivity of A2780/CP cells. We found that following VPA treatment, the expression of H19 and EZH2 decreased, but the expression of p21 and PTEN increased significantly. To investigate the involvement of H19 in VPA-induced apoptosis and cisplatin sensitivity, H19 was inhibited by a specific siRNA. Our results demonstrate that H19 knockdown by siRNA induced apoptosis and sensitized the A2780/CP cells to cisplatin-induced cytotoxicity. These data indicated that VPA negatively regulates the expression of H19 in ovarian cancer cells, which subsequently leads to apoptosis induction, cell proliferation inhibition, and overwhelming to cisplatin resistance. The implication of H19→EZH2→p21/PTEN pathway by VPA treatment suggests that we could repurpose an old drug, valproic acid, as an effective drug for treatment of ovarian cancer in the future.     

LIN28B confers radio-resistance through the posttranscriptional control of KRAS

To screen the differentially expressed microRNAs related to radio-resistance, we compared the microRNA profiles of lung cancer cells with different responses to ionizing radiation (IR). Of 328 microRNAs in microarray, 27 microRNAs were differentially expressed in NCI-H460 (H460) and NCI-H1299 (H1299) cells. Among them, let-7g was down-regulated in radio-resistant H1299 cells, and the level of let-7g was higher in radio-sensitive cells like Caski, H460, and ME180 in qRT-PCR analysis than in radio-resistant cells like A549, H1299, DLD1, and HeLa. Over-expression of let-7g in H1299 cells could suppress the translation of KRAS, and increase the sensitivity to IR. When we knockdown the expression of LIN28B, an upstream regulator of let-7g, the level of mature let-7g was increased in H1299 cells and the sensitivity to IR was also enhanced in LIN28B knockdown cells. From these data, we suggest that LIN28B plays an important role in radiation responses of lung cancer cells through inhibiting let-7g processing and increasing translation of KRAS.     

Engineered Bacteriorhodopsin May Induce Lung Cancer Cell Cycle Arrest and Suppress Their Proliferation and Migration

Highly expressible bacteriorhodopsin (HEBR) is a light-triggered protein (optogenetic protein) that has seven transmembrane regions with retinal bound as their chromophore to sense light. HEBR has controllable photochemical properties and regulates activity on proton pumping. In this study, we generated HEBR protein and incubated with lung cancer cell lines (A549 and H1299) to evaluate if there was a growth-inhibitory effect with or without light illumination. The data revealed that the HEBR protein suppressed cell proliferation and induced the G₀/G₁ cell cycle arrest without light illumination. Moreover, the migration abilities of A549 and H1299 cells were reduced by ~17% and ~31% after incubation with HEBR (40 μg/mL) for 4 h. The Snail-1 gene expression level of the A549 cells was significantly downregulated by ~50% after the treatment of HEBR. In addition, HEBR significantly inhibited the gene expression of Sox-2 and Oct-4 in H1299 cells. These results suggested that the HEBR protein may inhibit cell proliferation and cell cycle progression of lung cancer cells, reduce their migration activity, and suppress some stemness-related genes. These findings also suggested the potential of HEBR protein to regulate the growth and migration of tumor cells, which may offer the possibility for an anticancer drug.     

miR-9 and let-7g enhance the sensitivity to ionizing radiation by suppression of NFκB1

The activation of nuclear factor-kappa B1 (NFkB1) in cancer cells may confer resistance to ionizing radiation (IR). To enhance the therapeutic efficiency of IR in lung cancer, we screened for microRNAs (miRNAs) that suppress NFkB1 and observed their effects on radiosensitivity in a human lung cancer cell line. From time series data of miRNA expression in γ-irradiated H1299 human lung cancer cells, we found that the expression of miR-9 was inversely correlated with that of NFκB1. Overexpression of miR-9 down-regulated the level of NFκB1 in H1299 cells, and the surviving fraction of γ-irradiated cells was decreased. Interestingly, let-7g also suppressed the expression of NFκB1, although there was no canonical target site for let-7g in the NFκB1 3' untranslated region. From these results, we conclude that the expression of miR-9 and let-7g could enhance the efficiency of radiotherapy for lung cancer treatment through the inhibition of NFκB1.     

Distinguishable Targeting of Non-Small Cell Lung Cancer Using Hyaluronan Functionalized Platinum Nanoclusters and Their Inhibition Behaviors of Proliferation,Invasion, Migration

Lung cancer is the leading cause of cancer deaths worldwide and most cancer patients receiving conventional chemotherapy suffer from severe side effects due to the non-selective effects of chemotherapeutic drugs on normal cells. Targeted nanomaterials can obtain excellent accumulation at the tumor site through their active or passive targeting mechanisms, thereby reducing the toxicity of the drugs in various ways. In this study, hyaluronic acid (HA) which could specifically bind to CD44 on the surface of tumor cells, was used to modify amine-caged platinum nanoclusters (Pt NCs-NH₂) to obtain targeting HA-Pt NCs-NH₂. Based on the differential expression of CD44 on the surface of three lung cells (non-small cell lung cancer cell H1299, small cell lung cancer cell H446, and embryonic lung fibroblast HFL1), HA-Pt NCs-NH₂ can differentially enter the three cells and achieve their targeting of non-small cell lung cancer cell (NSCLC) cells. Pt NCs significantly inhibited the proliferation, migration and invasion of NSCLC cells and induced their apoptosis in comparison of classical cisplatin and carboplatin, showing a bright future in early diagnosis and treatment of NSCLC.     

HDAC Inhibitory and Anti-Cancer Activities of Curcumin and Curcumin Derivative CU17 against Human Lung Cancer A549 Cells

Previous research reported that the curcumin derivative (CU17) inhibited several cancer cell growths in vitro. However, its anticancer potential against human lung cancer cells (A549 cell lines) has not yet been evaluated. The purpose of this research was to examine the HDAC inhibitory and anti-cancer activities of CU17 compared to curcumin (CU) in A549 cells. An in vitro study showed that CU17 had greater HDAC inhibitory activity than CU. CU17 inhibited HDAC activity in a dose dependent manner with the half-maximal inhibitory concentration (IC₅₀) value of 0.30 ± 0.086 µg/mL against HDAC enzymes from HeLa nuclear extract. In addition, CU17 could bind at the active pockets of both human class I HDACs (HDAC1, 2, 3, and 8) and class II HDACs (HDAC4, 6, and 7) demonstrated by molecular docking studies, and caused hyperacetylation of histone H3 (Ac-H3) in A549 cells shown by Western blot analysis. MTT assay indicated that both CU and CU17 suppressed A549 cell growth in a dose- and time-dependent manner. Besides, CU and CU17 induced G2/M phase cell cycle arrest and p53-independent apoptosis in A549 cells. Both CU and CU17 down-regulated the expression of p53, p21, Bcl-2, and pERK1/2, but up-regulated Bax expression in this cell line. Although CU17 inhibited the growth of lung cancer cells less effectively than CU, it showed less toxicity than CU for non-cancer cells. Accordingly, CU17 is a promising agent for lung cancer treatment. Additionally, CU17 synergized the antiproliferative activity of Gem in A549 cells, indicating the possibility of employing CU17 as an adjuvant treatment to enhance the chemotherapeutic effect of Gem in lung cancer.     

Triptolide sensitizes lung cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by inhibition of NF-kappaB activation

TNF-related apoptosis-inducing ligand (TRAIL/Apo- 2L), a newly identified member of the TNF family promotes apoptosis by binding to the transmembrane receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). TRAIL known to activate NF-kappaB in number of tumor cells including A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells exerts relatively selective cytotoxic affects to the human tumor cell lines without much effect on the normal cells. We set out to identify an agent that would sensitize lung cancer cells to TRAIL-induced apoptosis through inhibition of NF-kappaB activation. We found that triptolide, an oxygenated diterpene extracted and purified from the Chinese herb Tripterygium wilfordii sensitized A549 and NCI-H1299 cells to TRAIL-induced apoptosis through inhibition of NF-kappaB activation. Pretreatment with MG132 which is a well-known NF-kappaB inhibitor by blocking degradation of IkappaBalpha also greatly sensitized lung cancer cells to TRAIL-induced apoptosis. Triptolide did not block DNA binding of NF-kappaB activated by TRAIL as in the case of TNF-alpha. It has been already proven that triptolide blocks transactivation of p65 which plays a key role in NF-kappaB activation. These observations suggest that triptolide may be a potentially useful drug to enhance TRAIL-induced tumor killing in lung cancer.     

Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells

Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the α subunit of Gs (GαsQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GαsQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GαsQL-stimulated Bak reporter luciferase activity. Expression of GαsQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Gαs activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Gαs augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.     

Low-dose radiation response of the p21WAF1/CIP1 gene promoter transduced by adeno-associated virus vector

In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (< 1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.     

Cancer cells undergoing epigenetic transition show short-term resistance and are transformed into cells with medium-term resistance by drug treatment

To elucidate the epigenetic mechanisms of drug resistance, epigenetically reprogrammed H460 cancer cells (R-H460) were established by the transient introduction of reprogramming factors. Then, the R-H460 cells were induced to differentiate by the withdrawal of stem cell media for various durations, which resulted in differentiated R-H460 cells (dR-H460). Notably, dR-H460 cells differentiated for 13 days (13dR-H460 cells) formed a significantly greater number of colonies showing drug resistance to both cisplatin and paclitaxel, whereas the dR-H460 cells differentiated for 40 days (40dR-H460 cells) lost drug resistance; this suggests that 13dR-cancer cells present short-term resistance (less than a month). Similarly, increased drug resistance to both cisplatin and paclitaxel was observed in another R-cancer cell model prepared from N87 cells. The resistant phenotype of the cisplatin-resistant (CR) colonies obtained through cisplatin treatment was maintained for 2–3 months after drug treatment, suggesting that drug treatment transforms cells with short-term resistance into cells with medium-term resistance. In single-cell analyses, heterogeneity was not found to increase in 13dR-H460 cells, suggesting that cancer cells with short-term resistance, rather than heterogeneous cells, may confer epigenetically driven drug resistance in our reprogrammed cancer model. The epigenetically driven short-term and medium-term drug resistance mechanisms could provide new cancer-fighting strategies involving the control of cancer cells during epigenetic transition.Subject terms: Tumour heterogeneity, Epigenetics     

Synthesis and antiproliferative evaluation of 2,3-diarylquinoline derivatives

A number of 2,3-diarylquinoline derivatives were synthesized and evaluated for antiproliferative activities against the growth of six cancer cell lines including human hepatocellular carcinoma (Hep G2 and Hep 3B), non-small cell lung cancer (A549 and H1299), and breast cancer (MCF-7 and MDA-MB-231) cell lines. The preliminary results indicated that 6-fluoro-2,3-bis{4-[2-(piperidin-1-yl)ethoxy]phenyl}quinoline (16b) was one of the most active compounds against the growth of Hep 3B, H1299, and MDA-MB-231 with a GI(50) value of 0.71, 1.46, and 0.72 μM respectively which was more active than tamoxifen. Further investigations have shown that 16b induced cell cycle arrest at G2/M phase followed by DNA fragmentation via an increase in the protein expression of Bad, Bax and decrease in Bcl-2, and PARP which consequently cause cell death.     

Gypensapogenin H from hydrolyzate of total Gynostemma pentaphyllum saponins induces apoptosis in human breast carcinoma cells

Abstract

Gypensapogenin H (Gyp H) is a novel dammarane-type triterpene, isolated from hydrolyzate of total saponins from Gynostemma pentaphyllum. Our previous work demonstrated that Gyp H exhibited potent growth inhibitory effects on tumor cells. It significantly inhibited the growth of human breast cancer cells (MDA-MB-231), while having low toxicity to normal human breast epithelial cells, MCF-10a. Further mechanistic study demonstrated that Gyp H decreased survival, inhibited proliferation, migration, induced apoptosis and led to cell cycle arrest. For the MDA-MB-231 cell lines, Gyp H increased expression of P21, Bax and cytochrome c, induced PARP cleavage and activated caspases. Gyp H also reduced expression of CDK2/4, CyclinD1, E2F1 and Bcl2, which associated with the cell cycle arrest. Thus, our finding may be useful for understanding the mechanism of action of Gyp H on breast cancer cells and suggest that Gyp H would be a leading agent for the treatment of breast cancer.     

p53 overexpression represses androgen-mediated induction of NKX3.1 in a prostate cancer cell line

Prostate cancer is a disease involving complicated multiple-gene alterations. Both NKX3.1 and p53 are related to prostate cancer and play crucial roles in prostate cancer progression. However, little is known about the relationships and interactions between p53 and NKX3.1 in prostate cancer. We found that NKX3.1 expression is down-regulated by over-expression of wild type (wt) p53 in prostate cancer LNCaP cells. NKX3.1 is down-regulated at both the mRNA and protein levels by p53 over- expression due to either transient transfection of exogenous p53 or induction of endogenous p53. p53 over-expression represses androgen-induced transactivation of NKX3.1 by inhibiting the promoter of the androgen acceptor (AR) gene and by blocking AR-DNA binding activity. In addition, transfection with the p21 expression vector (pPSA-p21) showed that p21 does not reduce NKX3.1 expression, indicating that NKX3.1 expression is not the result of nonspecific effects of cell growth arrest. Our results provide biochemical and cellular biologic evidence that NKX3.1 is down-regulated by p53 over-expression in prostate cancer cells.     

Cytotoxic properties of the nitrosyl iron complex with phenylthiyl

Nitrosyl compounds based on the iron-sulfur clusters are a promising class of inorganic nitric oxide donors. We studied cytotoxic properties of one of such NO donors, viz., the nitrosyl [2Fe-2S] complex with phenyl ligands (Ph-complex). The Ph-complex induces the HeLa and H1299 tumor cell death. The Ph-complex and cisplatin used in combination exhibit synergistic cytotoxicity. During studies of the poly(ADP-ribose) polymerase degradation, it was found that the HeLa cell death induced by the Ph-complex proceeds presumably by the mechanism of apoptosis. In the MCF7 cells, the Ph-complex causes induction of p53 protein expression and changes in its apparent molecular weight.     

Insulin Receptor Substrate 1 Is Involved in the Phycocyanin-Mediated Antineoplastic Function of Non-Small Cell Lung Cancer Cells

Phycocyanin, derived from marine algae, is known to have noteworthy antineoplastic properties. However, the underlying mechanism involved in phycocyanin-mediated anti-growth function on non-small cell lung cancer (NSCLC) cells is still ambiguous. Here, we investigated the mechanism of action of phycocyanin on H1299, A549, and LTEP-a2 cells. According to the results obtained, insulin receptor substrate 1 (IRS-1) expression was reduced by phycocyanin. Cell phenotype tests showed that siRNA knockdown of IRS-1 expression significantly inhibited the growth, migration, colony formation, but promoted the apoptosis of NSCLC cells. Meanwhile, phycocyanin and IRS-1 siRNA treatment both reduced the PI3K-AKT activities in NSCLC cells. Moreover, overexpression of IRS-1 accelerated the proliferation, colony formation, and migration rate of H1299, A549, and LTEP-a2 cells, which was contradicting to the knockdown results. Overall, this study uncovered a regulatory mechanism by which phycocyanin inhibited the growth of NSCLC cells via IRS-1/AKT pathway, laying the foundation for the potential target treatment of NSCLC.     

Cranberry proanthocyanidins mediate growth arrest of lung cancer cells through modulation of gene expression and rapid induction of apoptosis

Cranberries are rich in bioactive constituents purported to enhance immune function, improve urinary tract health, reduce cardiovascular disease and more recently, inhibit cancer in preclinical models. However, identification of the cranberry constituents with the strongest cancer inhibitory potential and the mechanism associated with cancer inhibition by cranberries remains to be elucidated. This study investigated the ability of a proanthocyanidin rich cranberry fraction (PAC) to alter gene expression, induce apoptosis and impact the cell cycle machinery of human NCI-H460 lung cancer cells. Lung cancer is the leading cause of cancer-related deaths in the United States and five year survival rates remain poor at 16%. Thus, assessing potential inhibitors of lung cancer-linked signaling pathways is an active area of investigation.     

Malformin-A1 (MA1) Sensitizes Chemoresistant Ovarian Cancer Cells to Cisplatin-Induced Apoptosis

High-grade epithelial ovarian cancer is a fatal disease in women frequently associated with drug resistance and poor outcomes. We previously demonstrated that a marine-derived compound MalforminA1 (MA1) was cytotoxic for the breast cancer cell line MCF-7. In this study, we aimed to examine the effect of MA1 on human ovarian cancer cells. The potential cytotoxicity of MA1was tested on cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) ovarian cancer cell lines using AlamarBlue assay, Hoechst dye, flow cytometry, Western blot, and RT-qPCR. MA1 had higher cytotoxic activity on A2780S (IC50 = 0.23 µM) and A2780CP (IC50 = 0.34 µM) cell lines when compared to cisplatin (IC50 = 31.4 µM and 76.9 µM, respectively). Flow cytometry analysis confirmed the cytotoxic effect of MA1. The synergistic effect of the two drugs was obvious, since only 13% of A2780S and 7% of A2780CP cells remained alive after 24 h of treatment with both MA1 and cisplatin. Moreover, we examined the expression of bcl2, p53, caspase3/9 genes at RNA and protein levels using RT-qPCR and Western blot, respectively, to figure out the cell death mechanism induced by MA1. A significant down-regulation in bcl2 and p53 genes was observed in treated cells compared to non-treated cells (p < 0.05), suggesting that MA1 may not follow the canonical pathway to induce apoptosis in ovarian cancer cell lines. MalforminA1 showed promising anticancer activity by inducing cytotoxicity in cisplatin-sensitive and cisplatin-resistant cancer cell lines. Interestingly, a synergistic effect was observed when MA1 was combined with cisplatin, leading to it overcoming its resistance to cisplatin.     

Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line

Neuronal ubiquitin C-terminal hydrolase (UCH-L1) has been linked to Parkinson's disease (PD), the progression of certain nonneuronal tumors, and neuropathic pain. Certain lung tumor-derived cell lines express UCH-L1 but it is not expressed in normal lung tissue, suggesting that this enzyme plays a role in tumor progression, either as a trigger or as a response. Small-molecule inhibitors of UCH-L1 would be helpful in distinguishing between these scenarios. By utilizing high-throughput screening (HTS) to find inhibitors and traditional medicinal chemistry to optimize their affinity and specificity, we have identified a class of isatin O-acyl oximes that selectively inhibit UCH-L1 as compared to its systemic isoform, UCH-L3. Three representatives of this class (30, 50, 51) have IC(50) values of 0.80-0.94 micro M for UCH-L1 and 17-25 micro M for UCH-L3. The K(i) of 30 toward UCH-L1 is 0.40 micro M and inhibition is reversible, competitive, and active site directed. Two isatin oxime inhibitors increased proliferation of the H1299 lung tumor cell line but had no effect on a lung tumor line that does not express UCH-L1. Inhibition of UCH-L1 expression in the H1299 cell line using RNAi had a similar proproliferative effect, suggesting that the UCH-L1 enzymatic activity is antiproliferative and that UCH-L1 expression may be a response to tumor growth. The molecular mechanism of this response remains to be determined.