Cytotoxic Effects Of Chemotherapeutic Agents Research Articles

Nitric Oxide Reverts the Resistance to Doxorubicin in Human Colon Cancer Cells by Inhibiting the Drug Efflux

Multidrug resistance (MDR) is a phenomenon by which cancer cells evade the cytotoxic effects of chemotherapeutic agents. It may occur through different mechanisms, but it often correlates with the overexpression of integral membrane transporters, such as P-glycoprotein (Pgp) and MDR-associated proteins (MRPs), with resulting decrease of drug accumulation and cellular death. Doxorubicin is a substrate of Pgp; it has been suggested that its ability to induce synthesis of nitric oxide (NO) could explain, at least in part, its cytotoxic effects. Culturing the human epithelial colon cell line HT29 in the presence of doxorubicin, we obtained a doxorubicin-resistant (HT29-dx) cell population: these cells accumulated less intracellular doxorubicin, were less sensitive to the cytotoxic effects of doxorubicin and cisplatin, overexpressed Pgp and MRP3, and exhibited a lower NO production (both under basal conditions and after doxorubicin stimulation). The resistance to doxorubicin could be reversed when HT29-dx cells were incubated with inducers of NO synthesis (cytokines mix, atorvastatin). Some NO donors increased the drug accumulation in HT29-dx cells in a guarosine-3':5'-cyclic monophosphate-independent way; this effect was associated with a marked reduction of doxorubicin efflux rate in HT29 and HT29-dx cells, and tyrosine nitration in the MRP3 protein. Our results suggest that onset of MDR and impairment of NO synthesis are related; this finding could point to a new strategy to reverse doxorubicin resistance in human cancer.

2-(8-Hydroxy-6-methoxy-1-oxo-1Η-2-benzopyran-3-yl)propionic Acid, a Small Molecule Isocoumarin, Potentiates Dexamethasone-Induced Apoptosis of Human Multiple Myeloma Cells

2-(8-Hydroxy-6-methoxy-1-oxo-1Eta-2-benzopyran-3-yl)propionic acid (NM-3) is a small molecule isocoumarin derivative that has recently entered clinical trials as an orally bioavailable anticancer agent. NM-3 induces lethality of human carcinoma cells by both apoptotic and nonapoptotic mechanisms and potentiates the effects of cytotoxic chemotherapeutic agents. The present studies have evaluated the effects of NM-3 on human multiple myeloma (MM) cells. The results demonstrate that NM-3 potentiates dexamethasone-induced killing of both dexamethasone-sensitive MM1.S and dexamethasone-resistant RPMI8226 and U266 MM cells. We show that NM-3 enhances dexamethasone-induced release of the mitochondrial apoptogenic factors cytochrome c and Smac/DIABLO. The results also demonstrate that NM-3 enhances dexamethasone-induced activation of the intrinsic caspase-9->caspase-3 apoptotic pathway. In concert with these results, NM-3 potentiates dexamethasone-induced apoptosis of MM1.S cells. Moreover, NM-3 acts synergistically with dexamethasone in inducing apoptosis of the dexamethasone-resistant RPMI8226 and U266 MM cells. These findings indicate that NM-3 may be effective in combination with dexamethasone in the treatment of MM.

The in vitro synergistic cytotoxic effects of inductive, concurrent and sequential cyclooxygenase-2 inhibitor with cisplatin against A549 cell line

9693 Background: Cyclooxygenase (COX)-2 inhibitors have been shown to exert antitumor activity against human lung cancer cell lines in vitro, and suppressive effect on lung tumors implanted into recipient mice, but concurrent use of COX-2 inhibitors and antineoplastic agents has not been proven clinically to be superior to chemotherapeutic agent alone. Sequential use of COX-2 inhibitors with chemotherapeutic agent might be a possible treatment option. We initiate in vitro study on the growth-inhibitory effect of inductive, concurrent and sequential celecoxib with cisplatin against A549 cell line to determine the most effective dose and schedule for maximal synergistic cytotoxic effects. We anticipate further appropriate treatment strategies to enhance the cytotoxic effect of chemotherapeutic agents using COX-2 inhibitors in the treatment of lung cancer. Methods: A549 cells were incubated with various concentration and combination of celecoxib and cisplatin for 4 days: celecoxib 24 hours prior to cisplatin as induction, celecoxib simultaneous with cisplatin as concurrent, and celecoxib 24 hours subsequent to cisplatin as sequential treatment. Time of exposure to cisplatin for all combination was 72 hours. Cell viability was determined using MTT assay. Results: The IC50 of cisplatin and celecoxib alone to A549 cell line was 16.64μM/l and 90.42μM/l, respectively. IC50 of cisplatin with celecoxib at 1.55μM/l for induction and concurrent treatment was 23.31μM/l, and 16.52μM/l, respectively, which was not superior to cisplatin alone while that of sequential treatment was 9.98μM/l, which showed synergistic cytotoxic effect against A549 cell line. Conclusions: Sequential celecoxib subsequent to cisplatin exerted the most potent in vitro synergistic cytotoxic effect against lung cancer cells. These may provide a rationale for future clinical trial to evaluate the effect of incorporating COX-2 inhibitors into the cisplatin-based regimen for the treatment of lung cancer. No significant financial relationships to disclose.

The in vitro synergistic cytotoxic effects of inductive, concurrent and sequential cyclooxygenase-2 inhibitor with cisplatin against A549 cell line

9693 Background: Cyclooxygenase (COX)-2 inhibitors have been shown to exert antitumor activity against human lung cancer cell lines in vitro, and suppressive effect on lung tumors implanted into recipient mice, but concurrent use of COX-2 inhibitors and antineoplastic agents has not been proven clinically to be superior to chemotherapeutic agent alone. Sequential use of COX-2 inhibitors with chemotherapeutic agent might be a possible treatment option. We initiate in vitro study on the growth-inhibitory effect of inductive, concurrent and sequential celecoxib with cisplatin against A549 cell line to determine the most effective dose and schedule for maximal synergistic cytotoxic effects. We anticipate further appropriate treatment strategies to enhance the cytotoxic effect of chemotherapeutic agents using COX-2 inhibitors in the treatment of lung cancer. Methods: A549 cells were incubated with various concentration and combination of celecoxib and cisplatin for 4 days: celecoxib 24 hours prior to cisplatin as induction, celecoxib simultaneous with cisplatin as concurrent, and celecoxib 24 hours subsequent to cisplatin as sequential treatment. Time of exposure to cisplatin for all combination was 72 hours. Cell viability was determined using MTT assay. Results: The IC50 of cisplatin and celecoxib alone to A549 cell line was 16.64μM/l and 90.42μM/l, respectively. IC50 of cisplatin with celecoxib at 1.55μM/l for induction and concurrent treatment was 23.31μM/l, and 16.52μM/l, respectively, which was not superior to cisplatin alone while that of sequential treatment was 9.98μM/l, which showed synergistic cytotoxic effect against A549 cell line. Conclusions: Sequential celecoxib subsequent to cisplatin exerted the most potent in vitro synergistic cytotoxic effect against lung cancer cells. These may provide a rationale for future clinical trial to evaluate the effect of incorporating COX-2 inhibitors into the cisplatin-based regimen for the treatment of lung cancer. No significant financial relationships to disclose.

Immunoprotection by botanical drugs in cancer chemotherapy

Most of the synthetic chemotherapeutic agents available today are immunosuppressants, cytotoxic, and exert variety of side effects that are particularly evident in cancer chemotherapy. Botanical based immunomodulators are often employed as supportive or adjuvant therapy to overcome the undesired effects of cytotoxic chemotherapeutic agents and to restore normal health. Total extract, polar and non-polar extracts, and their formulations, prepared from medicinal plants mentioned in Ayurveda, namely, Withania somnifera (Linn Dunal) (Solanaceae), Tinospora cordifolia (Miers) (Menispermaceae), and Asparagus racemosus (Willd.) (Liliaceae), exhibited various immunopharmacological activities in cyclophosphamide (CP)-treated mouse ascitic sarcoma. Treatment of ascitic sarcoma-bearing mice with a formulation of total extracts of Withania somnifera and Tinospora cordifolia (80:20) and alkaloid-free polar fraction of Withania somnifera resulted in protection towards CP-induced myelo- and immunoprotection as evident by significant increase in white cell counts and hemagglutinating and hemolytic antibody titers. Treatment with these candidate drugs will be important in development of supportive treatment with cancer chemotherapy.

Integrin signaling inhibits paclitaxel-induced apoptosis in breast cancer cells.

Inherent or acquired drug resistance is one of the major problems in chemotherapy. The mechanisms by which cancer cells survive and escape the cytotoxic effects of chemotherapeutic agents are essentially unknown. In the present study, we demonstrate that in the MDA-MB-231 and MDA-MB-435 breast cancer cells, ligation of beta1 integrins by their extracellular matrix ligands inhibits significantly apoptosis induced by paclitaxel and vincristine, two microtubule-directed chemotherapeutic agents that are widely used in the therapy of breast cancer. We show that beta1 integrin signaling inhibits drug-induced apoptosis by inhibiting the release of cytochrome c from the mitochondria in response to drug treatment. Further, integrin-mediated protection from drug-induced apoptosis and inhibition of cytochrome c release are dependent on the activation of the PI 3-kinase/Akt pathway. Our results identify beta1 integrin signaling as an important survival pathway in drug-induced apoptosis in breast cancer cells and suggest that activation of this pathway may contribute to the generation of drug resistance.

MDM2 Mediated Nuclear Exclusion of p53 Attenuates Etoposide-Induced Apoptosis in Neuroblastoma Cells

The p53 gene in neuroblastoma tumors (NB) is rarely mutated but the protein accumulates in the cytoplasm. Because p53 can mediate the cytotoxic effects of chemotherapeutic agents, it is important to determine whether accumulation of p53 in the cytoplasm impairs p53 function. Data presented here indicate that hyperactive nuclear export of p53 suppresses etoposide-induced apoptosis but does not prevent growth arrest. We compared p53 function in a pair of NB subclones. Our data show etoposide induces complete trans-location of p53 to the nucleus and activation of apoptosis in the neuroblastic NB cell line SH-SY5Y (N-type), which expresses low levels of MDM2. However, in Schwann cell-like SH-EP1 cells (S-type), which have up to 10-fold higher levels of MDM2, p53 accumulates in the cytoplasm and the cells are extremely resistant to etoposide-induced apoptosis. Notably, when MDM2 expression is inhibited in S-type cells, with a phosphorothioated antisense oligonucleotide (AS5), then p53 accumulates in the nucleus and the SH-EP1 cells undergo apoptosis. Surprisingly, induction of p21 and G1-arrest are not attenuated in S-type cells, despite the predominantly cytoplasmic location of p53. Whereas, G1-arrest is attenuated in the SH-SY5Y cells, which have high levels of nuclear p53. Taken together, these findings suggest attenuation of G1-arrest is related to the differentiation status of neuroblastomas and occurs downstream of p53 nuclear accumulation. These results demonstrate for the first time that hyperactive nuclear export of p53 attenuates chemotherapy-induced apoptosis in NB cells, and our findings suggest that inhibitors of MDM2 may enhance the therapeutic efficacy of etoposide by promoting apoptosis rather than trans-differentiation.

Insulin, chemotherapy, and the mechanisms of malignancy: the design and the demise of cancer

The endogenous molecular biology of cancer cells involves autocrine and paracrine secretion of insulin and insulin-like growth-factors I and II, which subserve energy production and growth stimulation, respectively, in these cells. These activities confer on cancer its malignant potential, working as they do autonomously, free from higher levels of integrated control. Taking advantage of cancer’s mechanisms of malignancy by employing exogenous insulin as a biologic response modifier, it is possible to potentiate the cytotoxic effects of chemotherapeutic agents for improved treatment of cancer. A synergy between certain membrane and metabolic effects of insulin on cancer cell molecular biology increases anticancer drug efficacy, and it does so with reduced doses of the drugs, enhancing their safety. This treatment strategy has been applied abroad over the last five decades with very promising clinical results.

Enhancement of antitumor effect of bleomycin by low-voltage in vivo electroporation: a study of human uterine leiomyosarcomas in nude mice.

Uterine leiomyosarcoma is an extremely malignant neoplasm with high rates of distant metastasis, and systemic chemotherapy is not particularly effective. Thus, the introduction of more active anticancer agents, or of a new drug delivery system, is urgently needed. Recently, electrochemotherapy has been introduced as a way of enhancing the cytotoxic effects of chemotherapeutic agents. This involves administering the drug in combination with electric pulses (which permeabilize tumor cell membranes and allow the drug to enter the cells). In particular, bleomycin (BLM) cannot cross the plasma membrane efficiently, but its cytotoxicity can be enhanced by electropermeabilization. The aim of the present study was to investigate the effect of low-voltage electroporation (EP) in combination with local BLM injection on the growth of uterine leiomyosarcoma in nude mice. Human uterine leiomyosarcoma cells (SK-LMS-1) were implanted subcutaneously into nude mice. Tumor growth in mice treated with EP (100 V/cm) plus BLM was compared with that in mice receiving BLM alone, EP alone, or no treatment (controls). Tissue BLM concentrations and histological analysis (including mitotic counts) were evaluated in tumor tissues. There was a significant reduction in tumor growth in mice that received EP with BLM. One hour after the treatment, the local BLM concentration was 10 times higher in the tumors that received EP with BLM than in those receiving only BLM. Moreover, the mitotic count was lower in the tumors that received EP plus BLM than in the controls. These results demonstrate the possible therapeutic value of low-voltage EP with BLM in human uterine leiomyosarcoma.

Amelioration of the cytotoxic effects of chemotherapeutic agents by grape seed proanthocyanidin extract.

Anticancer chemotherapeutic agents are effective in inhibiting growth of cancer cells in vitro and in vivo, however, toxicity to normal cells is a major problem. In this study, we assessed the effect of a novel IH636 grape seed proanthocyanidin extract (GSPE) to ameliorate chemotherapy-induced toxic effects in cultured Chang epithelial cells, established from nonmalignant human tissue. These cells were treated in vitro with idarubicin (Ida) (30 nM) or 4-hydroxyperoxycyclophosphamide (4HC) (1 microg/ml) with or without GSPE (25 microg/ml). The cells were grown in vitro and the growth rate of the cells was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; thiazolyl blue] assay. Our results showed that GSPE decreased the growth inhibitory and cytotoxic effects of Ida as well as 4HC on Chang epithelial cells in vitro. Because these chemotherapeutic agents are known to induce apoptosis in the target cells, we analyzed the Chang epithelial cells for apoptotic cell population by flow cytometry. There was a significant decrease in the number of cells undergoing apoptosis following treatment with GSPE. We also found increased expression of the anti-apoptotic protein Bcl-2 in GSPE-treated cells using western blot techniques. Thus, these results indicate that GSPE can be a potential candidate to ameliorate the toxic effects associated with chemotherapeutic agents and one of the mechanisms of action of GSPE includes upregulation of Bcl-2 expression.

Effects of cytotoxic chemotherapeutic agents on split-dose repair in intestinal crypt cells

Many cancer chemotherapeutic agents interact with radiation to enhance the amount of radiation damage observed in both tumor and normal tissues. It is important to predict this interaction and to determine the effect of drug on sublethal damage repair. To evaluate for effects in rapid renewing normal tissues, the intestinal crypt cell in vivo assay is an excellent one to employ. These studies investigate the effect of eleven cancer chemotherapeutic drugs on split-dose repair in the intestinal crypt cell of the mouse. LAF1 male mice, age 10-12 weeks, were exposed to whole-body irradiation with orthovoltage x-rays delivered as a single dose or as equally divided doses delivered with intervals between the two exposures of 2 to 24 h. In the experimental group, the cancer chemotherapeutic agent was administered intraperitoneally 2 h before the first radiation dose. At 3.6 days after the second irradiation, the mice were sacrificed; the jejunum was removed, fixed, and sectioned for light microscopy. The number of regenerating crypts were counted and corrected to represent the number of surviving cells per circumference. Of the eleven drugs tested, only carmustine eliminated split-dose repair. Cisplatin delayed repair, and methotrexate caused marked synchronization obliterating the observation of split-dose repair. Most cytotoxic chemotherapeutic agents do not inhibit sublethal damage repair in intestinal crypt cells when given 2 h before the first radiation exposure. Absence of the initial increase in survival seen with split-dose radiation is noted with carmustine and high-dose methotrexate.

Combination chemoimmunotherapy with interferon-alpha and cisplatin in patients with advanced non-small cell lung cancer

Non-small cell lung cancer (NSCLC) has a relatively low response rate to systemic chemotherapy. Recently, several investigations have shown that interferon may augment the cytotoxic effect of chemotherapeutic agents and may improve the result of chemotherapy in treating cancers.An open-label noncomparative phase II study investigated the efficacy and safety of combined chemoimmunotherapy for NSCLC using IFN-alpha, given by intramuscular injection with 9 million units thrice a week for a maximum of 26 weeks, and cisplatin by intravenous infusion, 100 mg/m2, every 4 weeks for a maximum of 6 cycles. Ten patients, with histology-confirmed NSCLC at stage IV of their diseases were enrolled. They included 5 men and 5 women, with an average age of 53.7 year. Five of them had adenocarcinoma; the other five had squamous cell carcinoma.Five patients, including two patients with squamous cell carcinoma and three with adenocarcinoma, obtained a partial response (response rate 50%). The time needed to achieve a sustained response ranged from 5 to 13 weeks with a median of 8 weeks. The response duration ranged from 14 to 37 weeks with a median of 20 weeks. The overall survival time for the ten patients ranged from 2 to 20 months with a median of 8 months. All 10 patients needed dose modifications of IFN-alpha and/or cisplatin because of myelosuppression and/or impaired renal function. Other frequently encountered side effects included gastrointestinal disturbances and a flu-like syndrome, but these were well tolerated.These preliminary results indicate that chemoimmunotherapy with IFN-alpha and cisplatin can be an effective alternative therapy for patients with advanced NSCLC, but there are significant side effects.

MULTIPLE PATHWAY DRUG-RESISTANCE (REVIEW)

During the past few years it has become apparent that multiple mechanisms of resistance are playing a role in the clinical manifestations of drug resistance. In different organs and tumors, various components of the resistance phenotype are expressed to varying degrees. The cells utilize multiple pathways to overcome the cytotoxic effects of chemotherapeutic agents. No single mechanism can account for the full extent of resistance.

Pathohistologische Serienschnittuntersuchungen bei Patienten mit kompletter Tumorremission nach durchgeführter zytostatischer Induktionstherapie

Initial combination drug regimen containing cis-platin in patients with head and neck cancer produced a high percentage of clinical response. Despite numerous trials showing high tumor response rates, a survival advantage resulting from the use of chemotherapy has yet not been demonstrated conclusively. This trial was initiated to study the cytotoxic effect of chemotherapeutic agents on squamous cell carcinomas. 31 patients with oropharyngeal carcinomas received prior to surgical resection a polychemotherapy consisting of cis-platin and bleomycin. After surgical tumor resection the histopathologic examination based on the serial sections of the tumor tissues and neck dissection blocks showed no case of complete tumor response. Even in 9 patients with complete clinical tumor response the histopathological investigation revealed tumor cells.

Short-term quantitative in vitro cytotoxicity assay involving an S-9 activating system

Mouse 3T3 fibroblasts grown in 96-well microtiter plates are used for a test which incorporates an S-9 mixed function oxidase system into a neutral red viability assay for the assessment of the acute cytotoxicity of xenobiotics in vitro. This sensitive, quantitative, semi-automated assay was suitable for the rapid screening of a broad spectrum of substances, including pharmaceuticals, carcinogens, and anti-neoplastic agents. The test was applicable to the analysis of toxic ranges, for the detection of biotransformability of parent compounds and for the evaluation of the cytotoxic effects of chemotherapeutic agents.

The use of an image analyser in human tumour clonogenic assays.

The counting of tumoral colonies growing in semisolid media must be achieved in human tumour clonogenic assays. An image analyser can be advantageously used to overcome the limit of the eye when performing this task. Colonies growing from human cancer cell lines were analysed. Quantitative results on the cytotoxic effect of chemotherapeutic agents were obtained that could not be reached by means of the eye.

The influence of agarose concentration on the cloning efficiency of a series of established human cell lines

The colony-forming efficiency in soft agarose of human tumour cells from five established lines may be increased (2–10 fold) by a reduction of the final agarose concentration from 0.3% to 0.16%. This increased efficiency is beneficial when evaluating cytotoxic effects of chemotherapeutic agents.