Effects of standard chemotherapy on tumor growth and regulation of multidrug resistance genes and proteins in childhood rhabdomyosarcoma

Abstract

The prognosis of rhabdomyosarcoma (RMS) in advanced stages is still sobering. Therapy is limited due to local tumor recurrence, development of metastases and multidrug resistance. The aim of this study was to investigate the development of multidrug resistance in cell lines and in xenografts of alveolar and embryonal RMS treated according to the German Soft Tissue Sarcoma Study (CWS). Alveolar and embryonal RMS cell lines were treated with Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide. Expression levels of resistance-associated genes were assessed using Real time-PCR. Nude mice (NMRI nu/nu, n = 10 per group) underwent xenotransplantation of human embryonal or alveolar RMS. Animals were treated with standard chemotherapeutic drugs Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide according to treatment schedules of the CWS-study. Tumor sizes were measured and relative tumor volumes were calculated. Animals were sacrificed after 20 days and standard histology, Real-time-PCR for MDR1-, MRP-, LRP- and MDM2-gene as well as immunohistochemistry for MDR1-, LRP-, and MRP-protein were performed. In the cell lines, an up-regulation of MDR-1 gene was found in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, an up-regulation of LRP and MRP was found. Standard chemotherapy of alveolar rhabdomyosarcoma resulted in a significant reduction of tumor growth (P < 0.05) in all groups. In embryonal rhabdomyosarcoma strongest effects were found after treatment with Ifosfamide, Vincristine and Carboplatin (P < 0.05). RT-PCR revealed a MDR1-dependent mechanism in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, MDR1 occurred to a lower degree. Immunohistochemistry revealed correlating expression levels of multidrug resistance-associated proteins. The use of established chemotherapy on human RMS in vivo had strong effects on xenografts compared to their controls. In all cases, there was only a reduction of tumor growth, but not a complete eradication of the tumors. Chemotherapy seemed to upregulate the expression of resistance-associated genes in vitro and in vivo. The mechanism of multidrug resistance depends on the tumor subtype. Therefore, further investigations will be required to evaluate multidrug resistance in patients and to investigate new modalities for a reversal of multidrug resistance.

Inhibition of glutathione-S-transferase as a treatment strategy for multidrug resistance in childhood rhabdomyosarcoma

“…Therefore, we employed an animal model of xenotransplanted RMS treated with vincristine. The effectiveness of chemotherapy with vincristine in RMS has been demonstrated previously by our group (10). Despite the long treatment of the tumors, we detected vital tumor cells after chemotherapy, indicating a selection of multidrug-resistant tumor cells in this model.…”

“…After several cycles of chemotherapy multidrug resistance (MDR) may develop and reduces the effectiveness of the cytotoxic agents (2). We previously described that MDR plays a role in childhood RMS by known mechanisms such as P-glycoprotein in alveolar subtype and multidrug resistance-associated protein (MRP) and lung resistance-related protein (LRP) in embryonal subtype (10). Although these mechanisms might be responsible for MDR in rhabdomyosarcoma in part, a complete reversal of MDR could not be achieved by MDR inhibitors (18).…”

“…as single agent on day 1 and 2 according to the treatment protocol of the Cooperative Soft Tissue Sarcoma Study (CWS 2002-P) of the German Society of Pediatric Hematology and Oncology (GPOH). Vincristine was chosen due to its strong antitumor effects as well as induction of multidrug resistance as previously shown (10). It was given in equitoxic doses for mice.…”

“…Various resistance-associated genes and proteins have been identified in RMS (10). We have previously demonstrated that MDR mechanisms in RMS depend on the histological subtype.…”

“…In eRMS, mechanisms like multidrug resistanceassociated protein (MRP) or lung resistance-related protein (LRP) seems to play a role for multidrug resistance in vitro. In vivo, these xenobiotic pumps were of less importance (10). Therefore, MDR in childhood RMS is not completely understood and other mechanisms may also play a role in these patients.…”