Prabhakara S, Kalia V K
Department of Biophysics, National Institute of Mental Health & Neuro Sciences, Bangalore, India.
Indian J Med Res. 2008 Aug;128(2):140-8.
BACKGROUND & OBJECTIVE: Temozolomide (TMZ), a second generation alkylating drug, an effective cytotoxic agent as well as radiosensitizer for malignant brain tumours, has side effects like myelosuppression. Lonidamine (LND) increases the effectiveness of several experimental multiple chemotherapy protocols, without increasing bone marrow toxicities and is effective in brain tumour patients. The objective of the present studies was to investigate whether combining clinically relevant doses of LND and TMZ could increase the proliferation and radiation response of malignant human brain tumour cells in vitro.
A malignant human glioma (U373MG) cell line was used in these studies. TMZ (20, 40 or 60 microM) or LND (100, 150 or 200 microM), or the combination of both (20 and 100 microM, respectively) in 0.1 per cent dimethyl sulphoxide (DMSO) were added three days after setting up cultures, in six well plates (5 x 10(4) cells/ well). The effects of continuous treatment for two days on proliferation response and cytotoxicity were studied after trypsinization; by cell counts and the uptake of trypan blue dye (0.5%). For the study of radiation (60Co-Gamma-rays, 2 Gy) response, drugs were removed 4 h after irradiation and cultures were grown further in drug free, normal growth medium for another 20 h or 44 h.
Continuous presence of TMZ or LND for two days significantly inhibited cell proliferation in a concentration dependent manner. The frequencies of non viable cells increased significantly only at higher concentrations of LND. Combination of 20 microM TMZ with 100 microM LND had additive effects on proliferation response, without affecting cell viability. Short-term drug treatments without irradiation did not induce micronuclei formation. Cell proliferation and viability were also not affected. However, post-irradiation presence of either of these drugs for 4 h significantly reduced the proliferation response, 24 and 48 h after treatments. It was further inhibited by the combination treatment. On the contrary, radiation induced micronuclei formation was enhanced by either of the drugs; which was significantly increased by the combined treatment, 24 h as well as 48 h after irradiation. No effects on cell viability were observed, immediately after these treatments as well as at later time points.
INTERPRETATION & CONCLUSION: Our findings showed that combination of TMZ and LND at clinically achievable, low plasma concentrations could inhibit tumour growth, and lonidamine could reduce the dose of temozolomide required for radiosensitization of brain tumours.
替莫唑胺（TMZ）是第二代烷基化药物，是一种有效的细胞毒性药物，也是恶性脑肿瘤的放射增敏剂，但有骨髓抑制等副作用。氯尼达明（LND）可提高多种实验性联合化疗方案的疗效，且不增加骨髓毒性，对脑肿瘤患者有效。本研究的目的是探讨联合临床相关剂量的LND和TMZ是否能在体外增强人恶性脑肿瘤细胞的增殖及放射反应。
本研究采用人恶性胶质瘤（U373MG）细胞系。在培养建立三天后，于六孔板（每孔5×10⁴个细胞）中加入0.1%二甲基亚砜（DMSO）中的TMZ（20、40或60微摩尔）或LND（100、150或200微摩尔），或两者联合（分别为20和100微摩尔）。胰蛋白酶消化后，研究连续两天治疗对增殖反应和细胞毒性的影响；通过细胞计数和台盼蓝染料（0.5%）摄取进行检测。为研究放射（⁶⁰Co-γ射线，2 Gy）反应，照射后4小时去除药物，然后在无药物的正常生长培养基中继续培养20小时或44小时。
TMZ或LND连续存在两天以浓度依赖方式显著抑制细胞增殖。仅在较高浓度的LND时，非存活细胞频率显著增加。20微摩尔TMZ与100微摩尔LND联合对增殖反应有相加作用，且不影响细胞活力。未照射时的短期药物处理未诱导微核形成。细胞增殖和活力也未受影响。然而，照射后这两种药物中任一种存在4小时均显著降低处理后24小时和48小时的增殖反应。联合处理进一步抑制了增殖反应。相反，放射诱导的微核形成在两种药物作用下均增强；联合处理在照射后24小时和48小时显著增加了微核形成。这些处理后即刻及随后时间点均未观察到对细胞活力的影响。
我们的研究结果表明，在临床可达到的低血浆浓度下，TMZ与LND联合可抑制肿瘤生长，且氯尼达明可降低脑肿瘤放射增敏所需的替莫唑胺剂量。
