Apoptosis--Molecular aspects

A Down's Syndrome related Single Minded 2 gene (SIM2), previously known to be
associated with Trisomy 21 was predicted by bioinformatics to be colon cancer specific.
In previous work from the laboratory using a patient tissue repository, an isoform of this
gene, short form (SIM2-s) was shown to be colon cancer specific. Inhibition of SIM2-s
expression by antisense technology resulted in cancer-cell specific apoptosis within 24
hours. Microarray-based gene expression profiling of the antisense-treated colon cancer
cells provided a fingerprint of genes involving key cell cycle, apoptosis, DNA damage
and differentiation genes. Taking hints from the microarray database, experiments were
initiated to decipher the molecular mechanism underlying the cancer specific function of
the SIM2-s gene. Using an isogenic cell system, apoptosis was found to be dependent
on DNA damage and repair gene, GADD45-a. Further, key pathways including p38 MAP
kinase (MAPK) and specific caspases were essential for apoptosis. Programmed cell
death was not dependant on cell cycle and was preceded by the induction of terminal
differentiation. To clarify whether SIM2-s function is a critical determinant of differentiation, stable transfectants of SIM2-s were established in a murine adipocytic
cell line (3T3-L 1 ). SIM2-s overexpression caused a pronounced block of differentiation
of the pre-adipocytes into mature adipocytes. A study of the differentiation pathway in
3T3-L 1 cells suggested that this block occurs early on in the cascade. These results
supported the starting premise that SIM2-s is a critical mediator of cell differentiation. To
clarify whether the SIM2-s gene has transforming potential, the SIM2-s gene was
overexpressed in the NIH3T3 murine fibroblast cell line. The cells expressing the human
SIM2-s gene exhibited shorter doubling time, abrogation of growth serum requirement,
greater cell number at saturation density and focus formation. In vivo tumorigenicity
assays showed tumor formation with long latency. These results provide strong evidence
for the role of SIM2-s gene in tumor cell growth and differentiation, and validate drug
therapy use for the gene.

In spite the heavy investments in therapeutic research breast cancer still impacts the
lives of women globally. The projected incidence of new cases in USA for 2008 is 67,770,
with estimated 40,480 deaths. In this study, we investigated the therapeutic efficacy of
Cytoreg®-genistein combination treatment on MCF-7 human breast cancer cells. MCF-7
cells were treated with genistein and Cytoreg® single and combination treatments for 24-
48hr; and the chemosensitivity assessed using bioassays: Trypan Blue and MTT for cell
viability; Ethidium bromide/Rhodamine 123 to assess apoptosis induction; F AM PolyCaspase
binding assay for mechanism of action. The overall data indicated dose- and timedependent
cell death in the MCF-cells and that apoptosis was the major means of treatmentinduced
growth inhibition. There was evidence of Cytoreg®-induced autophagy in the cells.
The overall findings indicated that genistein-Cytoreg® combination was more efficacious
than either genistein or Cytoreg® alone. Cytoreg® enhanced the phytosensitivity of MCF-7
cells to genistein isoflavone.

A large number of metal-containing compounds show significant activity against cancer cells and incorporating a metal into a polymer offers several possible advantages. Compounds of the type R2SnCl2 (R = methyl, ethyl, propyl, butyl, t-butyl, octyl and phenyl) were tested for the ability to inhibit the growth of Balb 3T3 mouse fibroblast cells and CAOV3 human ovarian carcinoma cells. Polymers of 2-chloro-1,4-benzenediamine and the same organotin dichloride were synthesized and tested as well. For both monomers and polymers, the pattern of growth inhibition relative to the R group was butyl > propyl = t-butyl = octyl = phenyl > ethyl > methyl. This and other aspects of the structure-activity relationship of the monomers and polymers were examined.

Phytochemicals are biologically active secondary plant metabolites that could mimic biological activities. In this study genistein isoflavone, a phytochemical present in soy was investigated to determine its effect on the growth of human breast cancer cell line GI-101 and normal breast epithelial cells in vitro. The cells were exposed to varying concentrations of genistein isoflavone for 24 and 48 hour time periods and the effect was determined using post-treatment assays: MTT and Trypan Blue for cell viability; LDH assay for cytotoxicity; Rhodamine 123/Propidium Iodide and Ethidium Bromide/Acridine Orange assays for treatment-induced apoptosis and FAM Poly caspase assay for mechanism of action. The overall results revealed that genistein inhibited cell growth and proliferation through apoptosis in the cells in both time and dose-dependent manner. Normal breast epithelial cells were not significantly affected by genistein at the corresponding dosages. Based on the results obtained, it was concluded that genistein isoflavone could offer therapeutic efficacy in human breast carcinoma without significantly affecting the normal breast epithelial cells.

Phytochemicals are biologically active secondary plant metabolites that have been shown to exhibit anti-cancer activity. The dietary phytochemicals genistein isoflavone and beta-lapachone, were investigated to determine their effect on the growth of human prostate adenocarcinoma cells in vitro. The cells were exposed to varying concentrations of both phytochemicals in single and combination treatments for specified time periods and their effect was determined using post-treatment cell viability, treatment-induced apoptosis and cell signaling assays. The overall results revealed that both phytochemicals inhibited cell growth and proliferation and induced apoptosis in a dose-dependent manner for both single and combination treatments. However, combination treatments were not significantly more effective than single treatment with either drug. Both phytochemicals could therefore offer therapeutic efficacy in human prostate adenocarcinoma.

Recent advancement in chemotherapy has resulted in higher and longer survival rates of testicular cancer patients. However the use of chemotherapeutic agents are not without serious, sometimes fatal side effects. This study investigated the potential therapeutic efficacy of pomegranate extracts in testis cancer cells, GC1-spg, in vitro. A battery of assays was used to determine the chemosensitivity of GC1-spg cells to two pomegranate extracts, S (seed) and P (pericarp), in single and combination treatments: MTS and LDH to determine post-treatment survival rate (growth inhibition) and cytotoxicity respectively; Acridine Orange/Ethidium Bromide fluorescent dye to assess treatment-induced apoptosis/necrosis; Annexin V-FITC and TUNEL assays for early and late apoptosis respectively. Results from the obtained data indicated that both extracts have significant cytotoxic effect on testicular cancer cells (GC1-spg) in single and combination treatments. The data revealed a dose and time dependency of chemosensitivity to both extracts; and that apoptosis was the major mechanism treatment-induced cell death. Synergism was also indicated in growth inhibition by combination treatment. These findings offer strong justification for further studies with pomegranate as potential phytotherapy.

Harnessing the human genome using bioinformatics lead to the discovery of a highly cancer-selective gene, Single Minded 2 gene (SIM2). An isoform of the SIM2 gene, the short-form (SIM2-s), was shown to be specific to colon, pancreas, and prostate tumors. Antisense inhibition of SIM2-s in a colon carcinoma derived cell line (RKO) caused inhibition of gene expression, growth inhibition and apoptosis in vitro and in nude mice tumorigenicity models. To understand the mechanism of Sim2-s antisense, the antisense treated RKO colon cancer cells were monitored for genome wide expression using Affymetrix GeneChipRTM technology. A list of apoptosis related genes was generated using GeneSpringRTM software. Select GeneChip RTM output was validated by Quantitative RT-PCR. Relevance of a key gene, Growth arrest and DNA damage inducible (GADD45a), in the SIM2-s pathway was established. These results will provide a basis for the future experiments to understand the mechanism underlying Sim2-s activation in specific tumors.

The therapeutic potential of pomegranate and genistein on growth inhibition of human breast cancer cells was investigated. Methods. Cells (MCF-7) were initially cultured for 48 hr to achieve 80% confluence; and then exposed to the agents in single and combination treatments. Post-treatment analysis was done by using a series of bioassays, including LDH, MTS, AcrO-EthBr, Annexin-FITC and TUNEL assays for growth inhibition and apoptosis detection; and Caspase-3 and NQO1 for molecular pathways of apoptosis. Results. Pomegranate and genistein showed significant dose- and time-dependent cytotoxic and growth inhibition effects as well as apoptosis induction in MCF-7 cancer cells, with significantly higher ( P < 0.01) effects in the combination treatments than in the single treatments. Both drugs induced apoptosis through a caspase-mediated mechanism and independent of NQO1. Discussion and conclusions. Pomegranate and genistein inhibit the growth of MCF-7 breast cancer cells through induction of apoptosis with combination treatment being more efficacious than single treatments.