Ronaldo Ramos

Cell Cycle Perturbations of Platinum Derivatives on Two Ovarian Cancer Cell Lines

Cisplatin continues to be one of the most commonly used cytotoxic agent. Problems of drug resistance and nephrotoxicity have generated interest in new platinum derivatives. In this study, we used flow cytometry to study their effects on cell kinetics and to see if the extent of cell cycle perturbations can be used to determine relative potency. The following four platinum derivatives were tested: cisplatin, carboplatin, 254S, and NK121 on two human ovarian cancer cell lines: BG1 and CAOV3. Flow cytometric analysis revealed a dynamic spectrum of cell kinetic perturbations, which included sequential S-G2 block, concomitant S-G2 block, and a dominant S block with abolition of G2 block. Platinum derivatives NK121, 254S, and CARBO induced an average of 54.5 +/- 5.6, 21.2 +/- 5.5, and 2.5 +/- 2.8% more S-G2 blocks than cisplatin, respectively. When comparing the severity of S-G2 blocks and requiring a p-value of 0.05, the order of increasing potency was: cisplatin, carboplatin, 254S, and NK121.

In vitro evaluation of a novel chemotherapeutic agent, Adozelesin, in gynecologic-cancer cell lines

SummaryAdozelesin is a derivative of an extremely cytotoxic compound, CC1065. This entirely new class of drug binds preferentially to DNA and facilitates alkylation reaction. In the present study, we used the adenosine triphosphate (ATP) chemosensitivity assay to compare the cytotoxic potency of Adozelesin with that of common chemotherapeutic agents in ten gynecologic-cancer cell lines. Flow cytometry was also used to study its effects on cell-cycle kinetics. The mean drug concentrations required to produce a 50% reduction in ATP levels as compared with controls [IC50] were: Adriamycin, 0.17±0.06 μm; 4OH-Cytoxan, 18±3 μm; cisplatin, 17±7 μm; 5-fluorouracil, 183±116 μm; and Adozelesin, 11.0±5.4pm. Thus, Adozelesin was 104–107 times more potent than Adriamycin, cisplatin, 5-fluorouracil, and Cytoxan. Cell kinetics studies revealed significant S and G2 blocks such as those previously reported for other alkylating agents.

Determination of hormonal response in uterine cancer cell lines by the ATP bioluminescence assay and flow cytometry

Although progesterone receptor status has been shown to correlate with response to hormonal therapy, not all progesterone receptor-positive patients respond to this treatment and additional biologic assays are needed to help better predict clinical response to hormonal therapy. This study explored the potential of the ATP bioluminescence assay and flow cytometry as biological assays of hormonal response. Five uterine cancer cell lines were used: AE7, ECC-1, HEC1A, AN3, and SKUT1B. Cells were exposed to Provera or tamoxifen at 0.1, 0.2, 0.5, 1, 2, and 5X (X equal to peak plasma concentrations: 1.0 micrograms/ml Provera and 0.1 micrograms/ml tamoxifen). For correlation, estrogen and progesterone receptors were determined by the standard dextran-coated charcoal method. Only AE7 and ECC-1 were positive for progesterone receptors (501 fmol/mg AE7, 194 fmol/mg ECC-1) and the rest were negative (less than 8 fmol/mg). Tamoxifen exerted no inhibition to the above cell lines. Meanwhile, Provera exerted dose-response inhibition on both AE7 and ECC-1 cell lines. The effects of accumulation of G0-G1 phase and reduction of S, G2 cells (P less than 0.05), but not on the HEC1A cell line (P = 0.4). These changes confirmed the antiproliferative property of Provera. Further studies are needed to establish the role of the ATP bioluminescence assay and flow cytometry as biological assays of hormonal response.

The effects of provera on chemotherapy of uterine cancer cell lines

Even though progestin is commonly added to many chemotherapy regimens in the treatment of uterine cancers, its role is still unproven. Since progestin is antiproliferative, its tendency to arrest cells in G1 phase may interfere with cytotoxic mechanisms. The ATP chemosensitivity assay and flow cytometry were used to study the effects of a progestational compound such as Provera on three single agents and four drug combinations. Uterine cancer cell lines included progesterone-receptor (PR)-positive AE7 and ECC1 and PR-negative HEC1A, HEC1B, AN3, and SKUT1B. Provera selectively affected only PR-positive cell lines. It imposed an antiproliferative effect on drug-induced cell-cycle perturbations by reducing G2 and S blocks and minimizing G1 depletion. When using IC50s (concentrations required for 50% growth inhibition) of 0.5 as a cutoff for drug sensitivity and resistance, Provera significantly improved the IC50s of the drug-resistant subgroup from 1.95 +/- 0.36 to 0.71 +/- 0.19 (P = 0.009) but not those of the drug-sensitive subgroup (P = 0.13). In summary, Provera appeared to work independently from cytotoxic mechanisms. Its improvement of cytotoxicity was most pronounced in resistant cell lines bearing progesterone receptors.

Spectrum of cell-cycle kinetics of alkylating agent Adolezesin in gynecological cancer cell lines: correlation with drug-induced cytotoxicity

SummaryAdolezesin is an analog of CC-1065. These compounds are among the most potent alkylating agents known to date. Currently Adolezesin is undergoing phase I clinical trials at several cancer centers in the USA. While the cytotoxic effects of Adolezesin have been addressed elsewhere, its effects on cell-cycle kinetics have not been reported. Flow cytometry was performed on five human gynecological cancer cell lines: AN3, AE7, BG1, HEC1A, and SKUT1B. Exposure to Adolezesin (U73975, Upjohn Co.) was done at near confluency at 0, 0.1, 0.2, 0.5, 1 and 5x, withx=10 pg/ml as reference concentration, for 90 min. Cell samples were taken by trypsinization at 0, 24, 48, 72, 96, and 168 h for flow cytometry. The ATP chemosensitivity assays were performed on the above cell lines to establish dose/response curves. Flow-cytometric analyses revealed that there was a spectrum of cell-cycle perturbations, which included biphasic S and G2 blocks, reverse dose-dependent G2 blocks, and a sequential relationship of S and G2 blocks. This study demonstrated that the cell kinetic response to Adolezesin depended on several variables such as cell lines, drug sensitivity, concentrations, and sampling time. Because of this multivariable dependence and the lack of correlation with cytotoxicity, it would be difficult to use cell kinetic pertubations to predict chemotherapeutic response.

The use of ATP bioluminescence assay and flow cytometry in predicting radiosensitivity of uterine cancer cell lines: Correlation of radiotoxicity and cell cycle kinetics

Abstract Radiotherapy remains an important part of uterine cancer treatment. This study was designed to evaluate the potential of the ATP bioluminescence assay and flow cytometry for predicting radiosensitivity. Correlation of these two modalities revealed important insights into the relationship of radiotoxicity and cell kinetic effects. Six human uterine cancer cell lines were used: AE7, ECC1, HEC1A, HEC1B, AN3, and SKUT1B. Doses of cobalt 60 were 0, 1, 2, 5, 8, and 10 Gy. The ATP bioluminescence assays were performed on Day 7. Cell samples were taken at 0, 24, 48, 72, 96, and 168 hr for flow cytometry. The linear-quadratic model was used to fit survival data and mean inactivation dose D was calculated. Among parameters such as D, α and β coefficients, and surviving fraction at 2 Gy (SF2), both D and SF2 correlated best with survival data. Radiation effects on the cell cycle did not correlate with D and revealed two distinct patterns: either a G1 accumulation with mild G2 block or a G1 depletion and severe G2 block. The S cells consistently demonstrated a biphasic pattern with an initial reduction followed by an accumulation. In summary, the ATP assay was shown to have potential in the study of radiosensitivity. Radiation-induced cell kinetics appeared to vary with intrinsic cellular differences and, thus, could not be used to predict radiosensitivity.

Cell kinetic perturbations after irradiation and caffeine in the BG-1 ovarian carcinoma cell line

The treatment of ovarian carcinoma includes maximum surgical removal of the tumor tissue followed by irradiation or chemotherapy. In this study, the effects of caffeine on cell cycle traverse have been studied over a 168-hr period after X irradiation in BG-1 cells, an ovarian carcinoma cell line. The results were obtained with dual-parameter flow cytometric measurements of DNA and nuclear protein, using propidium iodide and fluorescein isothiocyanate. After radiation alone, a dose-related arrest of cells in G2 phase and cell kill were observed. Irradiating BG-1 cells with 5 Gy produced an accumulation of the cells in G2 at 24-72 hr postirradiation. When G2 was divided into low nuclear protein (G2A) and high nuclear protein (G2B) compartments, there was a G2A peak accumulation at 24 hr and a G2B peak accumulation at 48-72 hr. The addition of 1 mM caffeine to the culture media, starting immediately postirradiation, prevented G2 arrest, promoting a rapid traverse of cells through G2A to G2B to G1, which was associated with diminished survival.

Comparative evaluation of pirarubicin and adriamycin in gynecologic cancer cell lines

Abstract Pirarubicin (PIRA) has been shown to have improved potency with less cardiac toxicity in several phase I and II clinical trials in Japan and Europe. Since Adriamycin (DXR) remains one of the most potent drugs in treatment of gynecologic cancers, this derivative has the potential to become an important chemotherapeutic agent. In this study, we compared the performance of these two drugs against a panel of 10 gynecologic cancer cell lines. The ATP chemosensitivity assays were used to determine dose-response curves. Flow cytometry was used to study cell kinetic response to both drugs. Using an IC50 value of 0.2 μg/ml as a cutoff for drug sensitivity, 4 cell lines, ECC1, HEC1B, BG1, and SKOV3, were considered resistant to DXR. By comparing IC50s, PIRA was 3.4 ± 0.4 times more potent than DXR ( P = 0.05). The other 6 cell lines, AN3, AE7, HEC1A, CAOV3, SKUT1B, and ME180, were considered sensitive to DXR. In this group of cell lines, PIRA was 1.6 ± 0.3 times more potent than DXR ( P = 0.5). Both PIRA and DXR elicited a spectrum of cell kinetics. By comparing the magnitude of G2 blocks at 0.1 μg/ml, PIRA was approximately 2–5 times more potent than DXR in SKUT1B, HEC1A, and BG1 cell lines. PIRA also displayed a reverse dose-response pattern of G2 block so that at high dose, cell cycle kinetics would mirror those of untreated controls. This observation supports the presence of a resistant tumor subpopulation and the concept of tumor heterogeneity.