CytoregR inhibits growth and proliferation of human adenocarcinoma cells via induction of apoptosis

Abstract

Background: Cancer is one of the devastating neovascular diseases that incapacitate so many people the world over. Recent reports from the National Cancer Institute indicate some significant gain therapy and cancer management as seen in the increase in the 5-year survival rate over the past two decades. Although near-perfect cure rate have been reported in the early-stage disease, these data reveal high recurrence rate and serious side effects including second malignancies and fatalities. Most of the currently used anticancer agents are only effective against proliferating cancer cells. Thus attention has been focused on potential anti-cancer agents capable of killing cancer cells independent of the cell cycle state, to ensure effective elimination of most cancer cells. The objective of this study was to test the chemosensitivity and potential mechanism of action of a novel cancer drug, CytoregR, in a panel of human cancer cells.

Methods: the study was performed using a series of bioassays including Trypan blue exclusion, MTS Growth inhibition, LDH-cytotoxicity, TUNEL-Terminal DNA fragmentation Apoptosis Assay, and the Caspase protease CPP32 activity assays.

Results: CytoregR induced significant dose- and time-dependent inhibition of growth in all the cells; with significant differences in chemosensitivity (P < 0.05) between the target cells becoming more apparent at 48 hr exposure. CytoregR showed no significant effect on normal cells relative to the tumor cells. Growth inhibition in all the cells was due to induction of apoptosis at lower concentrations of cytoregR (> 1:300). CytoregR-induced caspase protease-3 (CPP32) activation significantly and positively correlated with apoptosis induction and growth inhibition; thus implicating CPP32 as the principal death pathway in cytoregR-induced apoptosis.

Conclusion: CytoregR exerted a dose-and time-dependent growth inhibitory effect in all the target cells through induction of apoptosis via the CPP32 death pathway, independent of hormonal sensitivity of the cells. The present data indicate that not only could CPP32 provide a potential target for regulation of cytoregR-induced apoptosis but also that cytoregR could play a significant role in chemotherapeutic regimen in many human malignant tumors.

Figure 1

Growth inhibition of human adenocarcinoma cells. Cells were grown and treated with indicated concentrations of cytoregR as described in the methods. Cell growth inhibition (cell death) was assessed by passage of Trypan blue dye into the cells, observed under microscope. The percentage of the live cells was determined by as described in the methods. A-24 hr: Percentage of live cells after 24 hr treatment. B-48 hr: The percentage of live cells at 48 hr treatment. The values represent the means +SEM of triplicates from two independent experiments. Bars = SEM

Figure 2

Growth inhibition of human adenocarcinoma cells. Cells were grown and treated with cytoregR for up to 48 hr as described in the methods Cell viability was assessed by the ability of live cells to metabolically reduce MTS Cell viability was assessed by the ability of live cells to metabolicallyreduce MTS to purple formazan after treatment with cytoregR for 48 hr. Percentage viability was determined by setting the absorbance of control, non-treated cells as 100%. Values represent the means + SEM of triplicates from two independent experiments Bars = SEM

Figure 3

Growth inhibition of human adenocarcinoma cells exposed to cytoregR. Cells were grown and treated with indicate concentrations/dilutions of cytoregR as described in themethods. Treatment-induced cytotoxicity was assessed by measuring the release of lactate dehydrogenase from dead cells, using the LDH assay as described in the methods. The values/data points represent the means ± SEM of triplicates from two independent experiments

Figure 4

Treatment-induced apoptosis in human adenocarcinoma cells Cells were grown and treated with indicate concentrations/dilutions of cytoregR as described in the methods. Percentage of apoptosis was determined by the TUNEL DNA fragmentation assay as described in the methods. The values represent the means ± SEM of triplicates from two independent experiments.

Figure 5

Correlation of % viable cells as assessed by the Trypan blue exclusion assay and apoptosis in treated cells. Percentage apoptotic cells was determined by the TUNEL DNA fragmentation assay as described. The treatment-induced reduction in viable cells corresponded with simultaneous increase in apoptotic cells at corresponding dilutions/concentrations of cytoregR. The data points represent the means ± SEM of triplicates from two independent experiments.

Figure 7

Photomicrographic changes in human adenocarcinoma cells, LNCaP, characteristic of apoptosis; becoming more evident after 48 hr treatment with 1:500 dilution of cytoregR. A: control LNCaP cells without treatment and incubatingfor 48 hr; cells showing normal morphological characteristics of proliferation. B: Light photomicrograph of LNCaP cells after 48 hr treatment; showing cell shrinkage, nuclear condensation and fragmentation -classical characteristics of apoptosis.

Figure 6

Correlation of cytoregR-induced expression of caspase-3 protease (CPP32) and apoptosis in adenocarcinoma cells. Cells were grown and treated with varying dilutions of cytoregR as described in the methods. Caspase-3 protease activation was determined by using a fluorescent substrate for caspase-like proteases as described in the methods. CPP32 expression correlated positively with percentage of apoptosis induction. Data points represent the means ± SEM of triplicates from two independent experiments.