Kang Fang

Goniothalamin Inhibits Growth of Human Lung Cancer Cells through DNA Damage, Apoptosis, and Reduced Migration Ability

We evaluated the possible anticancer performance of a natural compound, goniothalamin (GTN), against human lung cancer using as a non-small cell lung cancer (NSCLC) cell line, H1299, as the model system. Cellular proliferation was significantly inhibited by GTN. Using an improved alkaline comet-nuclear extract (comet-NE) assay, GTN was found to induce a significant increase in the tail DNA. Wound healing and zymography assays showed that GTN attenuated cell migration and caused a reduction in the activity level of two major migration-associated matrix metalloproteinases, MMP-2 and MMP-9. It can be concluded that the DNA-damaging effect of GTN against lung cancer cells leads to growth inhibition as well as a depression in migration ability. Therefore, GTN has potential as a chemotherapeutic agent against lung cancer.

Etoposide (VP-16) sensitizes p53-deficient human non-small cell lung cancer cells to caspase-7-mediated apoptosis.

Human non-small-cell-lung-cancer (NSCLC) cells of p53-null genotype were exposed to low-dosage topoisomearse II inhibitor etoposide (VP-16). The cellular proliferation rate could be effectively inhibited by VP-16 in dose-dependent manner. The effective drug concentration for growth inhibition could be as low as 0.5 μ M and the apoptotic phenotype became evident 48 h later. In H1299 cells, VP-16-induced cytotoxic effect was demonstrated associated with apoptosis that disappeared when restored with wild-type p53. Cell cycle analysis revealed that, upon VP-16 induction, cell death began with growth arrest by accumulating cells at the G2-M phase. The cells at sub-G1 phase increased at the expense of those at G2-M transition state. To assess the regulation of cell cycle modulators, western blot analysis of H1299 cell lysates showed the release of apoptosis initiator, cytochrome c and apaf-1 hours following drug induction. The cleavage of downstream effectors, procaspase-9 and procaspase-7, but not procaspase-3, was accompanied with proteolysis of poly-(ADP-ribose) polymerase (PARP). VP-16-activated procaspase-7 cleavage was abrogated in cells with ectopically expressed p53.On the other hand, the inhibited procaspase-7 fragmentation by caspase-specific inhibitor reversed apoptotic phenotype caused by drug induction. Thus, VP-16-induced apoptotic cell death was contributed by caspase-7 activation inp53-deficient human NSCLC cells.

Cisplatin-induced senescence and growth inhibition in human non-small cell lung cancer cells with ectopic transfer of p16INK4a

DNA damage is lethal and capable of inducing cellular aging or apoptosis. In this work, the highly tumorigenic and cisplatin-resistant human non-small cell lung cancer (NSCLC) cells were transfected with construct encoding the complete sequence of p16INK4a (p16). The stable clones with elevated p16 exhibited enhanced sensitivities to low concentration cisplatin treatment. Further study indicated that cisplatin arrested cells at G2/M phase and the effectiveness is proportional to the level of p16 expressed. The growth of the xenograft tumors established by p16 transfectants in nude mice was also suppressed by cisplatin by inducing senescence-like phenotype. The data altogether indicated that, in cisplatin-resistant tumor cells with basal endogenous p16, the growth suppression by drugs can be greatly improved by ectopic gene transfer.

Transfection of anti-sense complementary DNA of human epidermal-growth- factor receptor attenuates the proliferation of human non-small-cell-lung- cancer cells

The proliferation of human non‐small‐cell‐lung‐cancer (NSCLC) cells is regulated by the epidermal‐growth‐factor‐receptor (EGFR)‐mediated autocrine loop that interacts with transforming growth factor‐α (TGF‐α) of autocrine or paracrine origin. We have shown that EGFR expression is elevated in the brain metastatic variant of human NSCLC cells H226Br, which thereby acquire their increased sensitivity toward exogenous TGF‐α. To determine detailed cell‐phenotype changes as a result of EGFR down‐regulation, H226Br cells were transfected with a human EGFR‐cDNA construct encompassing an N‐terminal fragment (1.8 kb) in anti‐sense orientation downstream of the cytomegalovirus (CMV) promoter. The EGFR transcript expressed in the 3′–5′ direction is expected to neutralize EGFR mRNA and to reduce protein expression correspondingly. The established cell lines resistant to G418 were shown integrated with the transfected construct and their proliferation rates reduced as compared with the parental cells and with those transfected with vector alone. Down‐regulated EGFR expression in cells with the anti‐sense construct can be confirmed by Scatchard analysis and suppressed EGFR kinase activity. The restrained‐growth phenotype is also demonstrated in the prolonged G2‐M phase during the cell cycle, and correlated with impairment of cell proliferation. This finding suggests that EGFR over‐expression is critical in maintaining the malignant phenotype of NSCLC cells, thereby providing a valuable biomarker and potential target prevention for lung‐cancer‐cell proliferation. Int. J. Cancer 81:471–478, 1999.

Additive effects of C2-ceramide on paclitaxel-induced premature senescence of human lung cancer cells

AIMS the aims of the study are to investigate the additive effect of exogenous short-carbon chain phospholipids, C(2)-ceramide, on an anti-cancer drug paclitaxel (PTX)-induced senescence of human non-small cell lung cancer (NSCLC) cells deficient in functional p53 and p16, and to examine whether mitogen-activated protein kinase (MAPK) plays a role in ceramide-sensitized senescence of NSCLC cells. MAIN METHODS to determine whether exogenous C(2)-ceramide renders lung cancer cells more sensitive to PTX treatment, techniques employing a flow cytometry-based cell cycle analysis and acidic β-galactosidase staining for senescent cells were used. Furthermore, to elucidate the role of MAPK proteins in modulating senescence, assays for protein levels of selective MAPKs and Bcl-2 family members, and detection of transcriptional levels senescence-associated genes were used in the study. KEY FINDINGS a sub-lethal dose of C(2)-ceramide sensitized the NSCLC H1299 cells to PTX treatment. The additive effects of C(2)-ceramide and PTX resulted in proliferative inhibition, G(2)-phase arrest of cell cycle, activation of p38 and eventually premature senescence. Importantly, neither p53, p21(waf1/cip1) nor p16(ink4) was shown to be involved in C(2)-ceramide-sensitized proliferative inhibition and senescence of H1299 cells by PTX in our study. SIGNIFICANCE our study demonstrates that the short-carbon chain C(2)-ceramide can effectively sensitize PTX-induced senescence of H1299 cells via both p21(waf1/cip1)- and p16(ink4)-independent pathways.

The aqueous extract of Prunella vulgaris suppresses cell invasion and migration in human liver cancer cells by attenuating matrix metalloproteinases.

The mechanism of action of Prunella vulgaris L. (PV) affecting cell migration and invasion of human liver cancer cells remains unknown. In this work we showed that the aqueous extract of PV affected migration and invasion of human liver carcinoma cells by inhibiting activities of metalloproteases, MMP-2 and MMP-9, without affecting cell viabilities. We further showed that PV suppressed migration through attenuation of enzymatic activities of MMP-9 and MMP-2 at transcriptional levels and the effects can be correlated with the status of p53 in hepatocarcinoma cells. This work provides a new dimension of understanding on Prunella vulgaris in restraining migration and invasion in human liver cancer cells.

Ectopic expression of herpes simplex virus‐thymidine kinase gene in human non‐small cell lung cancer cells conferred caspase‐activated apoptosis sensitized by ganciclovir

Human non‐small cell lung cancer (NSCLC) cells were transfected with recombinant prodrug herpes simplex virus type I thymidine kinase (HSV‐tk) cDNA, and the selected clones underwent apoptosis in response to induction by antiviral ganciclovir (GCV). The efficiency of GCV‐induced growth inhibition and the extent of the bystander effect were associated with the expression level of HSV‐TK in stable transfectants. Development in the HSV‐tk/GCV system toward cell death was initiated with cell‐cycle accumulation at S and G2/M phases, immediately followed by the appearance of sub‐G0/G1 cells after drug exposure. To investigate the regulation of cell‐cycle modulators during drug treatment, we analyzed release of the apoptosis initiator cytochrome c and activation of the downstream effectors caspase‐9, caspase‐3 and poly(ADP‐ribose)polymerase 16 hr after GCV sensitization, followed by transient escalation of tumor‐suppressor p53 and cell‐cycle modulators cyclin A and B1 before committing to programmed cell death. Furthermore, tumor regression was proportional to the degree of ectopic expression of the transferred HSV‐tk gene. Our results demonstrate that the HSV‐tk/GCV system effectively inhibits the proliferation of NSCLC cells in vitro and in vivo through potent induction of apoptosis, thus providing a rationale for further development.

An indolylquinoline derivative promotes apoptosis in human lung cancer cells by impairing mitochondrial functions

A number of effective anti-cancer drugs contain either indole or quinoline group. Compounds fused indole and quinoline moieties altogether as indolylquinoline were rarely reported as anti-cancer agents. We reported here that a synthetic indolylquinoline derivative, 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline (EMMQ), inhibited the growth of human non-small cell lung cancer (NSCLC) cells in dose- and time-dependent manners. The cytotoxicity was mediated through apoptotic cell death that began with mitochondrial membrane potential interruption and DNA damage. EMMQ caused transient elevation of p53 that assists in cytochrome c release, cleavage of downstream PARP and procaspase-3 and mitochondria-related apoptosis. The degree of apoptotic cell death depends on the status of tumor suppressor p53 of the target cells. H1299 cells with stable ectopic expression of p53 induced cytotoxicity by disrupting mitochondria functions that differed with those transfected with mutant p53. Knocking-down of p53 attenuated drug effects. EMMQ suppressed the growth of A549 tumor cells in xenograft tumors by exhibiting apoptosis characteristics. Given its small molecular weight acting as an effective p53 regulator in NSCLC cells, EMMQ could be an addition to the current list of lung cancer treatment.

Insulin-like growth factor-I is an autocrine regulator for the brain metastatic variant of a human non-small cell lung cell line

Insulin-like growth factor (IGF-I) is associated with autocrine and paracrine stimulation for cell growth and development of brain tumor cells. The function of IGF-I in the brain metastatic variant of human lung cancer cells is investigated. The cells used here were derived in vivo with intracarotid injection of human non-small cell lung carcinoma NCI-H226. The tumor was developed as a cultured cell line, H226Br. Unlike the parental cells, H226Br was tumorigenic in nu/nu nude mice. Reverse transcriptase-polymerase chain reaction showed that IGF-I transcript of H226Br is increased compared to that of parental cells. The amount of IGF-I secreted in cultured medium of H226Br is higher than that of cultured parental cells. The IGF-I receptor-specific antibody, alpha IR3, inhibits H226Br growth in serum-free culture. The results established that IGF-I is an autocrine growth regulator for human non-small cell lung cancer cells that progressed to brain.

Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53

In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells.