Enzymatic synthesis of perillyl alcohol derivatives and investigation of their antiproliferative activity

The monoterpene perillyl alcohol (POH), an intermediate in the plant terpenoid biosynthetic pathway, has well-established tumor chemopreventive and chemotherapeutic potential. We have previously shown that the primary hydroxyl group of POH is essential for its antitumor and anti-angiogenic activities. In the current study we present the enzymatic synthesis of two POH derivatives with different polar and hydrophobic characteristics, namely perillyl glucoside and perillyl glucoside fatty ester, through a two-step modification. Initial glucosylation of POH on its active hydroxyl group with D-(+)-glucose and subsequent esterification of the perillyl glucoside product with vinyl laurate were carried out using almond β-glucosidase and lipase B from Candida antarctica, respectively, in a low-water system. Optimization of enzymatic reactions was performed to achieve the highest possible conversion yields. The antitumor cell proliferation activity against mouse Lewis lung carcinoma cells was retained in both derivatives, although the perillyl glucoside ester showed greater inhibition than perillyl glucoside. Our results underline the feasibility of enzymatically producing novel bioactive analogs of phytochemicals displaying useful physicochemical properties.     

Dichloroacetate alters Warburg metabolism,inhibits cell growth,and increases the X-ray sensitivity of human A549 and H1299 NSC lung cancer cells

We investigated whether altering Warburg metabolism (aerobic glycolysis) by treatment with the metabolic agent dichloroacetate (DCA) could increase the X-ray-induced cell killing of the radiation-resistant human non-small-cell lung cancer (NSCLC) cell lines A549 and H1299. Treatment with 50 mM DCA decreased lactate production and glucose consumption in both A549 and H1299, clear indications of attenuated aerobic glycolysis. In addition, we found that DCA treatment also slowed cell growth, increased population-doubling time, and altered cell cycle distribution. Furthermore, we report that treatment with 50 mM DCA significantly increased single and fractionated X-ray-induced cell killing of A549 and H1299 cells. Assay of DNA double-strand break repair by neutral comet assays demonstrated that DCA inhibited both the fast and the slow kinetics of X-ray-induced DSB repair in both A549 and H1299 NSCL cancer cells. Taken together the data suggest a correlation between an attenuated aerobic glycolysis and enhanced cytotoxicity and radiation-induced cell killing in radiation-resistant NSCLC cells.     

Cytotoxicity and genotoxicity evaluation of antidote oxime HI-6 tested on eight cell lines of human and rodent origin

Oxime HI-6 is an efficient reactivator of the acetylcholinesterase inhibited by organophosphorous nerve agents. In this study we have estimated cytotoxicity of HI-6 by the colony forming assay and genotoxicity by the comet assay on human and rodent cell lines. IC50 of HI-6 assessed by the colony forming capacity was 3.59 mM for HeLa cells and 5.18 mM for a mouse cell line L929. Small difference in cytotoxicity was found among other cell lines tested: IC50 was 1.61 mM for human A549 cells, 1.14 mM for UROtse line, 1.96 mM and 1.71 mM for Chinese hamster cells AA8 and UV-20, respectively. The A549 cell viability measured with the MTT test was 5 times decreased comparing 2 and 24 hours of HI-6 oxime treatment. The 5 mM HI-6 concentration reduced the viability within 2 hours to 95% only, however, it induced a significant number of DNA breaks in mouse cells L929, and also in human UROtse and HepG2 cells. 1-β-D-arabinofuranosylcytosine (10(-4) M) and hydroxyurea (10(-2) M), supplemented to the cultivation medium, did not cause any significant accumulation of DNA breaks during treatment, which indicated that the nucleotide excision repair was not acting on the induced DNA damage.     

Differential effects of mesenchymal stem cells on a heterogeneous cell population within lung cancer cell lines

Although mesenchymal stem cells (MSCs) promote lung cancer growth in vivo, in vitro studies indicate that they inhibit the proliferation of lung cancer cells. Because malignant tumors contain a heterogeneous cell population with variable capacity for self-renewal, the aim of this study was to determine whether the inconsistencies between in vitro and in vivo studies are a result of differential effects of MSCs on the heterogeneous cell population within lung cancer cell lines. Human MSCs were isolated from the bone marrow, and their cell surface antigen expression and multi-lineage differentiation capacity was examined at passage 10. CD133+ cells were isolated from A549 and H446 cell lines using immunomagnetic separation. The effects of MSCs on the growth and microsphere formation of heterogeneous cell populations within two lung cancer cell lines (A549 and H446) were compared. MSCs inhibited the in vitro proliferation of both cell lines, but significantly accelerated tumor formation and stimulated tumor growth in vivo (P < 0.05). In CD133+ cells isolated from both A549 and H446 cells, co-culture with MSCs for 1–3 days significantly increased their proliferation (P < 0.05). MSCs also significantly increased microsphere formation in both cell lines (P < 0.05). Selective stimulation of CD133+ cell growth may account for the discrepant effects of MSCs on lung cancer progression.     

Perillyl alcohol inhibits TCR-mediated [Ca(2+)](i) signaling, alters cell shape and motility, and induces apoptosis in T lymphocytes

Perillyl alcohol (POH) inhibits isoprenylation and has shown anticancer and chemopreventive properties in rodent models. The mechanism that underlies the anticancer activity of POH and other isoprenylation inhibitors is unknown but has been postulated to involve decreased levels of isoprenylated Ras and Ras-related proteins. Previously we demonstrated that POH effectively inhibits human T cell proliferation in vitro and can prevent acute and chronic rejection in a rat cardiac transplant model. In this report, we investigate the effects of POH on T lymphocytes at the single-cell level. POH disrupts the polarized shape and motility of antigen-specific murine 1E5 T cells. Using an optical trap to position anti-CD3-coated beads in contact with 1E5 T cells, we demonstrate that POH inhibits their TCR-mediated calcium response. Furthermore, we show that POH preferentially induces apoptosis in PHA-activated human T cells as well as in 1E5 T cells.     

Effects of polyphyllin I on growth inhibition of human non-small lung cancer cells and in xenograft

Polyphyllin I (PPI), a small molecular monomer extracted from Rhizoma of Paris polyphyllin, shows strong anticancer effects in previous study. Human lung adenocarcinoma A549 cells, human lung squamous cell carcinoma SK-MES-1 cells, and human lung large cell carcinoma H460 cells were cultured and then treated with PPI. Cell proliferation and apoptosis were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, flow cytometry, western blot analysis, and DNA ladder. Athymic nude mice bearing tumors were injected with PPI, and tumor growth was recorded. Our results showed that PPI significantly inhibited the proliferation of three non-small cell lung cancer (NSCLC) cell lines, with the inhibitory concentrations (IC50) of 1.24, 2.40, and 2.33 μg/ml for A549, H460, and SK-MES-1 cells, respectively. After being treated with 2.5 μg/ml of PPI for 24 h, the apoptotic rate of A549 cells was 39.68%, which was remarkably higher than that of the control. Tumor growth was significantly inhibited in the PPI-treated group compared with the group treated with cisplatin (DDP) or PBS in the nude mice. PPI exhibits antitumor ability in NSCLC cells in vitro and in vivo, which might be related to the apoptosis induced by PPI.     

The mechanisms of nadroparin‐mediated inhibition of proliferation of two human lung cancer cell lines

Objectives

Clinical data suggest that heparin treatment improves survival of lung cancer patients, but the mechanisms involved are not fully understood. We investigated whether low molecular weight heparin nadroparin, directly affects lung cancer cell population growth in conventionally cultured cell lines.

Materials and methods

A549 and CALU1 cells’ viability was assessed by MTT and trypan blue exclusion assays. Cell proliferation was assessed using 5‐bromo‐2‐deoxyuridine incorporation. Apoptosis and cell‐cycle distribution were analysed by flow cytometry; cyclin B1, Cdk1, p‐Cdk1 Cdc25C, p‐Cdc25C and p21 expressions were analysed by western blotting. mRNA levels were analysed by real time RT‐PCR.

Results

Nadroparin inhibited cell proliferation by 30% in both cell lines; it affected the cell cycle in A549, but not in CALU‐1 cells, inducing arrest in the G₂/M phase. Nadroparin in A549 culture inhibited cyclin B1, Cdk1, Cdc25C and p‐Cdc25C, while levels of p‐Cdk1 were elevated; p21 expression was not altered. Dalteparin caused a similar reduction in A549 cell population growth; however, it did not alter cyclin B1 expression as expected, based on previous reports. Fondaparinux caused minimal inhibition of A549 cell population growth and no effect on either cell cycle or cyclin B1 expression.

Conclusions

Nadroparin inhibited proliferation of A549 cells by inducing G₂/M phase cell‐cycle arrest that was dependent on the Cdc25C pathway, whereas CALU‐1 cell proliferation was halted by as yet not elucidated modes.     

Perillyl alcohol and genistein differentially regulate PKB/Akt and 4E-BP1 phosphorylation as well as eIF4E/eIF4G interactions in human tumor cells

Previously we demonstrated that secondary products of plant mevalonate metabolism called isoprenoids attenuate 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA translational efficiency and cause tumor cell death. Here we compared effects of "pure" isoprenoids (perillyl alcohol and gamma-tocotrienol) and a "mixed" isoprenoid-genistein-on the PKB/Akt/mTOR pathway that controls mRNA translation and m(7)GpppX eIF4F cap binding complex formation. Effects were cell- and isoprenoid-specific. Perillyl alcohol and genistein suppressed 4E-BP1(Ser65) phosphorylation in prostate tumor cell lines, DU145 and PC-3, and in Caco2 adenocarcinoma cells. Suppressive effects were similar to or greater than that observed with a PI3 kinase inhibitor or rapamycin, an mTOR inhibitor. 4E-BP1(Thr37) phosphorylation was reduced by perillyl alcohol and genistein in DU145, but not in PC-3. Conversely, perillyl alcohol but not genistein decreased 4E-BP1(Thr37) phosphorylation in Caco2. PKB/Akt activation via Ser473 phosphorylation was enhanced in DU145 by perillyl alcohol and in PC-3 by gamma-tocotrienol, but was suppressed by genistein. Importantly, perillyl alcohol disrupted interactions between eIF4E and eIF4G, key components of eIF4F (m(7)GpppX) cap binding complex. These results demonstrate that "pure" isoprenoids and genistein differentially impact cap-dependent translation in tumor cell lines.     

Licarin A induces cell death by activation of autophagy and apoptosis in non-small cell lung cancer cells

Lung cancer has a relatively poor prognosis with a low survival rate and drugs that target other cell death mechanism like autophagy may help improving current therapeutic strategy. This study investigated the anti-proliferative effect of Licarin A (LCA) from Myristica fragrans in non-small cell lung cancer cell lines—A549, NCI-H23, NCI-H520 and NCI-H460. LCA inhibited proliferation of all the four cell lines in a dose and time dependent manner with minimum IC50 of 20.03?±?3.12, 22.19?±?1.37 µM in NCI-H23 and A549 cells respectively. Hence NCI-H23 and A549 cells were used to assess the ability LCA to induce autophagy and apoptosis. LCA treatment caused G1 arrest, increase in Beclin 1, LC3II levels and degradation of p62 indicating activation of autophagy in both NCI-H23 and A549 cells. In addition, LCA mediated apoptotic cell death was confirmed by MMP loss, increased ROS, cleaved PARP and decreased pro-caspase3. To understand the role of LCA induced autophagy and its association with apoptosis, cells were analysed following treatment with a late autophagy inhibitor-chloroquine and also after Beclin 1 siRNA transfection. Data indicated that inhibition of autophagy resulted in reduced anti-proliferative as well as pro-apoptotic ability of LCA. These findings confirmed that LCA brought about autophagy dependent apoptosis in non-small cell lung cancer cells and hence it may serve as a potential drug candidate for non-small cell lung cancer therapy.     

Chemotherapeutic effect of a novel temozolomide analog on nasopharyngeal carcinoma in vitro and in vivo

Background

Many patients with nasopharyngeal carcinoma (NPC) face poor prognosis. Due to its hidden anatomical location, the tumor is usually diagnosed quite late, and despite initially successful treatment with radiation and cisplatin, many patients will relapse and succumb to the disease. New treatment options are urgently needed. We have performed preclinical studies to evaluate the potential NPC therapeutic activity of a newly developed analog of temozolomide (TMZ), an alkylating agent that is the current chemotherapeutic standard of care for patients with malignant glioma.

Results

TMZ was covalently conjugated to the natural monoterpene perillyl alcohol (POH), creating the novel fusion compound NEO212. Its impact on two NPC cell lines was studied through colony formation assays, cell death ELISA, immunoblots, and in vivo testing in tumor-bearing mice. In vitro, NEO212 effectively triggered tumor cell death, and its potency was significantly greater than that of its individual components, TMZ or POH alone. Intriguingly, merely mixing TMZ with POH also was unable to achieve the superior potency of the conjugated compound NEO212. Treatment of NPC cells with NEO212 inactivated the chemoprotective DNA repair protein MGMT (O6-methylguanine methyltransferase), resulting in significant chemosensitization of cells to a second round of drug treatment. When tested in vivo, NEO212 reduced tumor growth in treated animals.

Conclusion

Our results demonstrate anticancer activity of NEO212 in preclinical NPC models, suggesting that this novel compound should be evaluated further for the treatment of patients with NPC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0175-6) contains supplementary material, which is available to authorized users.     

A specific isoform of nonmuscle myosin II-C is required for cytokinesis in a tumor cell line

Nonmuscle myosin IIs play an essential role during cytokinesis. Here, we explore the function of an alternatively spliced isoform of nonmuscle myosin heavy chain (NMHC) II-C, called NMHC II-C1, in the A549 human lung tumor cell line during cytokinesis. NMHC II-C1 contains an insert of 8 amino acids in the head region of NMHC II-C. First, we show that there is a marked increase in both the mRNA encoding NMHC II-C1 and protein in tumor cell lines compared with nontumor cell lines derived from the same tissue. Quantification of the amount of myosin II isoforms in the A549 cells shows that the amounts of NMHC II-A and II-C1 protein are about equal and substantially greater than NMHC II-B. Using specific siRNAs to decrease NMHC II-C1 in cultured A549 cells resulted in a 5.5-fold decrease in the number of cells at 120 h, whereas decreasing NMHC II-A with siRNA does not affect cell proliferation. This decreased proliferation can be rescued by reintroducing NMHC II-C1 but not NMHC II-A or II-B into A549 cells, although noninserted NMHC II-C does rescue to a limited extent. Time lapse video microscopy revealed that loss of NMHC II-C1 leads to a delay in cytokinesis and prolongs it from 2 to 8-10 h. These findings are consistent with the localization of NMHC II-C1 to the intercellular bridge that attaches the two dividing cells during the late phases of cytokinesis. The results suggest a specific function for NMHC II-C1 in cytokinesis in the A549 tumor cell line.     

Cytotoxicity and genotoxicity of zingiberene on different neuron cell lines in vitro

The main objective of this study is to investigate the cytotoxic, genotoxic and antioxidant properties of zingiberene (ZBN) in an in vitro rat brain cell culture study. The cytotoxic effect was determined against the rat neuron and N2a neuroblastoma (N2a-NB) cell lines using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while the antioxidant activity was assessed using the total antioxidant capacity (TAC) and total oxidative stress (TOS) assays. The effects on DNA damage were also evaluated in this study by the single cell gel electrophoresis assay. The results indicated that ZBN has an anti-proliferative activity suppressing the proliferation of N2a-NB cells at concentrations over 50 mg L⁻¹ and neuron cells at concentrations over 150 mg L⁻¹. In addition, ZBN treatments at higher doses (≤50 mg L⁻¹) led to increases of TOS levels in N2a-NB cell cultures. However 25 mg L⁻¹ of ZBN treatment caused increases of TAC levels in cultured neuron and N2a-NB cell cultures while ZBN at doses of 10–400 mg L⁻¹ did not increase the number of total damage score in both cell lines. This study clearly indicates that ZBN has a significant potential to be used as a natural anticancer agent in cultured N2a-NBs.     

In vitro biological evaluation of novel 7-O-dialkylaminoalkyl cytotoxic pectolinarigenin derivatives against a panel of human cancer cell lines

The effect of novel pectolinarigenin derivatives bearing a dialkylaminoalkyl substituent at O-7 on cell proliferation was evaluated in vitro in a panel of seven human cancer cell lines including renal adenocarcinoma ACHN, amelanotic melanoma C32, colorectal adenocarcinoma Caco-2, lung large cell carcinoma COR-L23, malignant melanoma A375, lung carcinoma A549 and hepatocellular carcinoma Huh-7D12 cell lines. Pectolinarigenin (2), obtained by hydrolysis of rutinose unit of the pectolinarin (1) isolated from Linaria reflexa, exhibited cytotoxic activity against Caco-2, A549 and A375 cell lines with IC(50) values of 5.3-8.2 microM. The most active pectolinarigenin derivative was 3 characterized by a dimethylamino-propoxy group in O-7 with IC(50) values of 7.2 and 7.4 microM against COR-L23 and A549 cell lines, respectively. A structure-activity relationship analysis of synthesized compounds was performed. None of the tested compounds affected the proliferation of skin fibroblasts 142BR suggesting a selective activity against tumor cells.     

Effects of coumarin and 7OH-coumarin on bcl-2 and Bax expression in two human lung cancer cell lines in vitro

Coumarin and its derivative 7-hydroxycoumarin (7-OHC) have antitumor and antimetastatic properties. The purpose of this study was to investigate the possible effects of these compounds on expression of the bcl-2 and Bax oncoproteins in two human lung cancer cell lines, A427 and Calu-1. The cells were cultured in vitro for 24 h in RPMI 1640 with 1.5% (v/v) ethanol, 1.0 mM ethanolic coumarin or 1.0 mM ethanolic 7-OHC. Viability was determined in each cell line by an MTT assay. Total protein was extracted from cell lysates and the bcl-2 and Bax oncoproteins were identified. Western blotting showed a decrease in bcl-2 and an increase in Bax in A427 cells cultured with coumarin or 7-OHC. Neither drug changed bcl-2 expression in Calu-1 cells compared to solvent controls, and Bax expression was only slightly increased by coumarin. We conclude that 7-OHC is a more potent inhibitor of cell proliferation than coumarin and has more marked effects on oncoprotein expression. Also, the A427 cell line was more sensitive to the drugs than Calu-1.     

Effect of tobacco extract on surfactant synthesis and its reversal by retinoic acid—role of cell–cell interactions in vitro

Tobacco induces oxidative stress in the alveolar epithelium and causes its damage. Retinoic acid (RA) has a cardinal role in alveolar cell growth, differentiation, and maturation. The aim of the study was to investigate the role of cell–cell interactions and whether RA could reverse the effect of tobacco extract on epithelial function as expressed by surfactant synthesis. For this, an in vitro model, which provides multiple cell type interactions, as seen in vivo, was used. We had used the major lung cell types, alveolar epithelial and mesenchymal cells represented by the cell lines A549 (human lung adenocarcinoma cell line), and human fetal lung fibroblast-1 (HFL-1) for developing the monoculture and co-culture systems and studied the effect of tobacco extract and retinoic acid. The effect of tobacco and retinoic acid both singly and in combination on proliferation and surfactant synthesis was analyzed. Retinoic acid induced proliferation and upregulated surfactant synthesis in monocultures and co-cultures. Tobacco extract at 100 μg/ml concentration decreased A549 proliferation and upregulated surfactant protein mRNA expression. In co-cultures treated with tobacco extract (100 μg/ml), retinoic acid (1 μM), regulated cell proliferation, and surfactant protein mRNA expression vis-à-vis the monoculture system. This clearly points to the fact that cell–cell interactions modulate the effect of additives or stimulants and help in assessing the in vivo combinatorial responses in vitro and that the retinoic acid effect is regenerative.     

Protein and m-RNA expression of farnesyl-transferases,RhoA and RhoB in rat liver hepatocytes: action of perillyl alcohol and vitamin A <Emphasis Type="BoldItalic">in vivo</Emphasis>.

Summary We analysed the action, in rats in vivo, of the protein isoprenylation inhibitor perillyl alcohol (POH) and that of vitamin A, alone or in association, on m-RNA and protein expression of farnesyltransferases (FTases α and β subunits) and their protein substrates RhoA and RhoB, in isolated hepatocytes. Combined administration of POH and vitamin A induced a sharp decrease in FTase α protein after 96 h, suggesting an involvement not only of farnesyltransferases but also of geranylgeranyltransferases, which share the FTase α protein. FTase β protein did not decrease. POH plus vitamin A, in contrast with POH or vitamin A alone, induced a decrease in RhoB protein, probably because of different cleavages. No modification was observed in RhoA protein. Vitamin A alone increased RhoB m-RNA and protein expression. As one of the functions of RhoB is cell polarisation, these data support our previous hypothesis of a polarised transport of vitamin A from hepatocytes to hepatic stellate cells. As the behaviours of m-RNAs and proteins in this study were often different, cytoplasmic metabolic pathways must be considered for the parameters studied. The behaviour of Rho B, which is thought to have an antioncogene function, is discussed in view of its isoprenylated forms in the membranes. These preliminary findings stress the need, when studying the association of two isoprenoids in cancer therapy, to consider normal as well as tumour-bearing animals.     

Polycystin‐1 affects cancer cell behaviour and interacts with mTOR and Jak signalling pathways in cancer cell lines

Polycystic Kidney Disease (PKD), which is attributable to mutations in the PKD1 and PKD2 genes encoding polycystin‐1 (PC1) and polycystin‐2 (PC2) respectively, shares common cellular defects with cancer, such as uncontrolled cell proliferation, abnormal differentiation and increased apoptosis. Interestingly, PC1 regulates many signalling pathways including Jak/STAT, mTOR, Wnt, AP‐1 and calcineurin‐NFAT which are also used by cancer cells for sending signals that will allow them to acquire and maintain malignant phenotypes. Nevertheless, the molecular relationship between polycystins and cancer is unknown. In this study, we investigated the role of PC1 in cancer biology using glioblastoma (GOS3), prostate (PC3), breast (MCF7), lung (A549) and colorectal (HT29) cancer cell lines. Our in vitro results propose that PC1 promotes cell migration in GOS3 cells and suppresses cell migration in A549 cells. In addition, PC1 enhances cell proliferation in GOS3 cells but inhibits it in MCF7, A549 and HT29 cells. We also found that PC1 up‐regulates mTOR signalling and down‐regulates Jak signalling in GOS3 cells, while it up‐regulates mTOR signalling in PC3 and HT29 cells. Together, our study suggests that PC1 modulates cell proliferation and migration and interacts with mTOR and Jak signalling pathways in different cancer cell lines. Understanding the molecular details of how polycystins are associated with cancer may lead to the identification of new players in this devastating disease.     

G2/M cell cycle arrest and induction of apoptosis by a stilbenoid, 3,4,5-trimethoxy-4'-bromo-cis-stilbene, in human lung cancer cells

Stilbenoids, including resveratrol (3,5,4'-trihydroxy-trans-stilbene) which is a naturally occurring phytoalexin abundant in grapes and several plants, have been shown to be active in inhibiting proliferation and inducing apoptosis in human cancer cell lines. Using resveratrol as the prototype, we have synthesized various analogs and evaluated their growth inhibitory effects in cultured human cancer cells. In the present study, we show that one of the stilbenoids, 3,4,5-trimethoxy-4'-bromo-cis-stilbene (BCS), was more effective than its corresponding trans-isomer and resveratrol on the inhibition of cancer cell growth. Prompted by the strong growth inhibitory activity of BCS (IC50; 0.03 microM) compared to its trans-isomer (IC50; 6.36 microM) and resveratrol (IC50; 33.0 microM) in cultured human lung cancer cells (A549), we investigated its mechanism of action. BCS induced arrest at the G2/M phase cell cycle in the early time and subsequently increased in the sub-G1 phase DNA contents in a time-dependent manner, indicating induction of apoptosis. Morphological observation with round-up shape and DNA fragmentation was also revealed the apoptotic phenomena. BCS treatment elevated the expression levels of the pro-apoptotic protein p53, the cyclin-dependent kinase inhibitor p21, and the release of cytochrome c in the cytosol. The down-regulation of checkpoint protein cyclin B1 by BCS was well correlated with the cell cycle arrest at G2/M. These data suggest the potential of BCS to serve as a cancer chemotherapeutic or chemopreventive agent by virtue of arresting the cell cycle and induction of apoptosis of human lung cancer cells.