cis-Pt Treatment Research Articles

EFFECT OF PROGESTERONE AND RU486 ON CISPLATINUM RESISTANCE IN OVARIAN CANCER CELLS

Epithelial ovarian cancer (EOC), the third most common cancer in women, has the highest mortality rate of any gynecological cancer. The primary chemotherapeutic treatment for EOC is one of the platinum drugs, such as carboplatinum or cisplatinum. Platinum drug treatment in combination with taxol is often more effective than platinum alone. However, development of resistance to the platinum drugs is common and leads to relapse of EOC. Therefore, it is important to find alternative treatments that can enhance the effectiveness of the platinum drugs and reverse resistance to platinum. Progesterone is a physiologically relevant hormone in the ovary and reported to increase apoptosis in epithelial cells, so we examined the effect of progesterone on the platinum sensitive and resistant cell lines OV2008 and C13, respectively. As with other steroids, progesterone regulates many cells via nuclear receptors and recent studies have indicated that steroid hormones may interact with the PI3- kinase pathway as well. Therefore, we also used RU486, a progesterone receptor antagonist and wortmannin, a PI3-kinase inhibitor. Treatment of OV2008 with 10, 20, 30, 50, or 100 μM cisplatinum (CisPt) caused a dosedependent decrease in cell viability (89 −24%). In contrast, even 100 μM CisPt only caused a decrease in viability to 83% in the C13 cells, confirming that this cell line is resistant to CisPt. In the next set of experiments, cells were treated with 8.0 μM progesterone, 8.0 μM RU486, or 1.0 μM wortmannin in the absence or presence of 30 μM CisPt. Experiments were repeated 3 to 14 times. Treatment of either OV2008 or C13 cells with progesterone or wortmannin had no effect on cell viability (90 −97 %), nor did progesterone or wortmannin alter the effect of 30 μM CisPt on cell viability in either cell line (65 −69 % or 88 −97 % for OV2008 or C13, respectively). Although RU486 alone significantly decreased cell viability in OV2008 to 78.3 ± 5.5 % and C13 to 80.4 ± 4.2 %, RU486 did not alter the effect of CisPt on either cell line. Interestingly, combining progesterone + RU486 further decreased the viability of OV2008 cells to 64.2 ± 8.2 %, but didn't enhance the CisPt effect (58.7 ± 5.6 %). Conversely, progesterone + RU486 without CisPt was no more effective at lowering viability in C13 cells than RU486 alone. However, treatment of C13 cells with CisPt and progesterone + RU486 significantly decreased cell viability to 59.2 ± 7.4 %, thus reversing the resistance to CisPt in these C13 cells. The effect of combining wortmannin + progesterone + RU486 wasn't significantly different from that of progesterone + RU486 without or with CisPt treatment. Thus, the progesterone + RU486 combination appears to significantly inhibit cell viability in OV2008 and to reverse the CisPt resistance in the C13 cells. It is not yet clear by what mechanism this agonist and its antagonist work together to produce these effects, but it doesn't appear to involve the PI3-kinase pathway. (Supported by an OBR Research Challenge Grant) (poster)

Effects of flavonoids on cisplatin-induced apoptosis of HL-60 and L1210 leukemia cells

Effects of three flavonoids, quercetin (QU), galangin (GA), and chrysin (ChR) on cisplatin (cis-Pt)-induced apoptosis of human promyelocytic leukemia HL-60 cells and murine leukemia L1210 cells were investigated. The quantitative analysis of apoptotic DNA fragmentation was used to show that preincubation of cells with flavonoids can influence cis-Pt-induced apoptosis in different way. ChR had no effect, QU enhanced, and GA reduced apoptotic DNA fragmentation. It is also shown that combined treatment with QU and cis-Pt showed synergistic effect, however, GA combined with cis-Pt exhibited antagonism on cytotoxicity in L1210 murine leukemia cells. We assume that tested flavonoids affect the important biological activities connected with cancer chemotherapy and chemoprevention as they differently modulated the sensitivity of cells to cis-Pt treatment. QU is presented as pro-apoptotic agent and GA as agent with anti-apoptotic potential.

Energy dispersive X-ray fluorescence as a tool in biomedical analysis

The energy dispersive X-ray fluorescence spectrometer (EDXRF) developped together with the Siemens Company (FRG) allows to perform rapid multi-elemental analysis of biological fluids without pretreatment of the samples. The performance of this technique and its features are reported in two different fields: blood analysis and pulmonary investigations. Blood and plasma analysis were performed for more than 2500 patients and a statistical treatment of the data lead to the normal concentrations of trace elements Se, Rb, Cu, Zn, Fe, Ca, Br, K. Toxic elements such as Hg or Pb and also drugs like Au and Pt compounds can be detected even after a long period after treatment. To illustrate the performance of the spectrometer we report the influence of Cis-Pt treatment on the trace element concentrations in plasma of patients with anemia. The EDXRF spectrometry was also used to investigate the inorganic composition of the broncho-alveolar lavage fluids. After determination of the normal concentration range, it was possible to characterize some typical pulmonary diseases due to occupational exposure. Some examples of detected pneumoconiosis and for some patients the presence in the alveoli of elements due to special medication (Amiodarone, Aurothiopropanol-sulfonate) are mentioned. Other fields are being investigated such as: follow-up of Mn dependent enzymes in neuronal and glial cells in culture; origin of lead in dental tissues or composition of lyophilized food powders. They will be briefly discussed.

Induction of umu gene expression by cross-links and other DNA lesions

The induction of umu gene expression by DNA cross-links was investigated in various strains of E. coli with different DNA-repair capacities. Expression was measured by quantifying enzymatic activity of β-galactosidase produced under regulation of the umu promoter carried on a plasmid carrying the umuC-lacZ gene fusion. The treatment with MMC induced gene expression more efficiently in a wild-type strain when compared with an excision-repair-deficient strain ( uvrA). In contrast, PUVA and cis-Pt treatment induced higher levels of the gene expression in the uvrA strain than in the wild-type strain, as did other DNA-damaging agents including 4NQO, MNNG and MMS. None of these chemicals induced umu expression in either lexA and recA strains. The mechanisms of the induction of umu expression by DNA cross-links in relation to DNA damage and repair are discussed.

Reinitiation at the λ DNA origin accompanies the host SOS response

Abnormal reinitiation of replication from λ origins has previously been found during infection in the presence of caffeine or cis-diamminedichloroplatinum II ( cis-Pt) or when λ infects a P2 lysogen. It was further shown that the reinitiations arising from cis-Pt treatment took place during the SOS response induced by the template damage caused by the drug. It is now shown that SOS induction by uv irradiation of the host also results in reinitiation events and that it is the SOS response itself rather than some other direct effect of the damaged host template that is responsible for the phenomenon. Parental sections of λ replicative intermediates can supercoil, whereas daughter segments cannot. To explain the control that prevents reinitiation, it is proposed that normally the origin sequence has to be under superhelical tension to be a suitable substrate for the initiating machinery; once a round is in progress, the daughter origin sequences would not be under such tension and would therefore be inactive. It is shown that in an SOS environment the proposed requirement for a superhelical origin sequence is relaxed and consequently the control against reinitiation lost. Under such conditions, primary growing points that have encountered template lesions terminate and a new wave of replication initiates.

Renal irradiation and the pharmacology and toxicity of methotrexate and cisplatinum

We used a rat model to study the effects of renal irradiation on the pharmacology of methotrexate (MTX) and cisplatinum (cis-Pt). Unanesthetized rats were given bilateral kidney irradiation (20 Gy in 9 fractions). At 9 months after irradiation, 3% of the animals had died and survivors showed moderately impaired renal function. At 15 months, 30% of the animals had died and survivors showed severely impaired renal function. Some animals were given i.v. MTX 1 week to 15 months after irradiation. In irradiated rats, the area under the NM plasma clearance curve equaled that of controls through 6 months, and was significantly above controls from 9 months on. Other animals were given i.p. cis-Pt 1 week to 9 months after irradiation. The acute toxicity of cis-Pt was the same in control and irradiated rats when cis-Pt was given immediately before or after irradiation. Beginning 3 months after irradiation there was a progressive increase in cis-Pt toxicity and a simultaneous decrease in urinary platinum excretion. Irradiated animals that survived cis-Pt treatment showed increased radiation nephritis; the greatest effect occurred when cis-Pt was given 3 months or more after irradiation. NM and cis-Pt clearance decreased when renal dysfunction was first observed and changes in renal function preceded changes in drug clearance and toxicity.

Reinitiation during λ DNA replication resulting from either cis-Pt treatment or infection of a P2 lysogenic strain

Nested areas of replication are observed in phage λ replicative intermediates and arise from reinitiation from the X origin. Reinitiation occurs when the first round of λ replication takes place in the presence of the drug cis-Pt or when λ infects a host which has been preincubated with the drug. In the latter case it is shown that the infection proceeds during the expression of SOS functions induced in the host as a result of the drug treatment. When λ infects a host lysogenic for phage P2, an interference process occurs which prevents formation of λ phage. The λ DNA does, however, undergo at least one round of replication but is abnormal in that λ origins reinitiate to form nested areas of replication similar to those resulting from exposure to the drug cis-Pt.

Host-cell reactivation of cis-diamminedichloroplatinum(II)-treated SV40 DNA in normal human, Fanconi anaemia and xeroderma pigmentosum fibroblasts

Cell survival after treatment with cis-diamminedichloroplatinum(II) [ cis-Pt(II)] and host-cell reactivation of cis-Pt(II)-treated SV40 DNA were investigated using two Fanconi anaemia, one xeroderma pigmentosum of complementation group A, and three normal human control fibroblast cell strains. The Fanconi anaemia and xeroderma pigmentosum cell strains showed an increased sensitivity to the cytotoxic action of cis-Pt(II) treatment, suggesting a deficiency in the repair pathway of cis-Pt(II)-induced damage. In addition, the survival of cis-Pt(II)-treated SV40 DNA was about 2-fold lower in xeroderma pigmentosum cells than in control cells. No difference in viral DNA survival was found between Fanconi anaemia and control cells, although the Fanconi anaemia cells were more sensitive to the cytotoxic action of treatment with cis-Pt(II) than the xeroderma pigmentosum cells in the clonogenic cell survival assay.

The nature of inactivating lesions produced by platinum (II) complexes in phage λ DNA

Lambda DNA loses transfectivity and acquires interstrand cross-links after treatment with either trans-Pt(II) or cis-Pt(II). With trans-Pt(II) there is close to an equivalence between the fraction of λ DNA cross-linked and the fraction inactivated. In contrast, with cis-Pt(II) there are approx. 5 inactivating lesions for each λ DNA interstrand cross-link. These results suggested that trans-Pt(II) does not introduce intrastrand inactivating lesions into λ DNA while cis-Pt(II) does so. To verify this conclusion, the cross-linked and uncross-linked fractions of λ DNA treated with trans-Pt(II) or cis-Pt(II) were separated on alkaline sucrose gradients. After trans-Pt(II) treatment, the uncross-linked fraction of λ DNA was transfective when renaturated. However after cis-Pt(II) treatment the uncross-linked fraction of λ DNA was not transfective when renatured. Thiourea treatment restored transfectivity to all inactivated fractions, showing that these lesions are reversible. We conclude that trans-Pt(II) inactivates λ DNA primarily by introducing interstrand cross-links but that cis-Pt(II), although it also introduces interstrand cross-links, inactivates λ DNA primarily by introducing intrastrand lesions.

Effects of cis-platinum(II) diamminedichloride on survival and the rate of DNA synthesis in synchronously growing Chinese hamster V79-379A cells in the absence and presence of caffeine inhibited post-replication repair; Evidence for an inducible repair mechanism

The mechanism by which cis-Platinum(II) diamminedichloride, ( cis-Pt(II)), inhibited DNA synthesis was investigated using synchronously growing Chinese hamster V79-379A cells. The progression of G 1 phase treated cells into S phase was not inhibited. However the rate of DNA synthesis throughout the S phase was depressed in a dose dependent manner compared to a control. The peak rate of DNA synthesis was delayed and the duration of the S phase was extended resulting in a dose dependent delay in the timing of mitosis in the first cell cycle after treatment. The delay in the timing of mitosis after the second S phase in treated cells was comparable to the delay observed after the first cycle. Post treatment incubation of cells in the presence of a non toxic concentration of caffeine resulted in an alteration in the pattern of DNA synthesis, leading to a reversal of the dose dependent delay in the timing of the peak rate of DNA synthesis to a position corresponding to the control. Caffeine potentiated the toxicity of cis-Pt(II) only if it was added during the first cell cycle after treatment. The patterns of DNA synthesis in synchronous cultures of Chinese hamster V79-379A cells after cis-Pt(II) treatment appeared to be similar to those previously demonstrated for difunctional alkylating agents. The results were discussed in relation to an existing model in which it was postulated that a DNA replication dependent repair mechanism enables cells to replicate DNA using a cis-Pt(II) damaged DNA template. This mechanism has been shown in Chinese hamster V79-379A cells to be sensitive to non toxic concentrations of caffeine resulting in an inhibition of the rate of ligation of nascent DNA which is followed by an increase in chromosome aberrations and cell death. The results presented support the proposed model and emphasise the importance of this replication repair mechanism in enabling cells to tolerate unexcised damage to their genome. In addition we propose that cis-Pt(II) damage stimulates the induction of a system which is involved in enhancing so called post replication repair in Chinese hamster V79 cells and that this induction might be inhibited by the presence of caffeine.

Cis—Dichlorodiammineplatinum (II) and its Effect on Microfilaments in Mammalian Cells

The regression of various tumors after cis-dichlorodiammineplatinum (II) (cis-Pt (II) treatment has been suggested to be mediated through mitotic inhibition and enhancement of the immune system. Present study illustrates the possible action of cis-Pt (II) on microfilaments and their role in the prevention of cytokinesis.Sarcoma-180 ascites cells in culture were treated with cis-Pt(II) (5ppm; 2 μg/ml) for intervals of 15, 30, 60 and 120 minutes. Controls were incubated in culture medium without cis-Pt (II) . Both the treated and non-treated cultures were then glycerinated and incubated with heavy meromyosin (HMM) (Ishikawa, et al., 1969. J. Cell Biol., 43, 312). Heavy meromyosin was prepared from myosin (Lowery, et al., 1969, J. Mol. Biol., 42, 1) using trypsin as inhibitor. Cell cultures treated with cis-Pt(II) for 30 minutes were also transferred to fresh medium containing 25μg/ml cytochalasin B and 1% dimethyl sulfoxide (DMSO) for 2-24 hours.