In vitro antineoplastic and anti-CSC effects of simvastatin in human acute lymphoblastic leukemia/lymphoma cell lines

RESUMO

INTRODUÇÃO: Acute lymphoblastic leukemias (ALL) are malignant disorders of immature B and T cells that occur characteristically in children, generally under the age of 6, representing 80% of childhood leukemias. Although therapeutic regimens for the pediatric age group have evolved in recent decades, in adulthood, the cure rate still remains close to 40%. The prognosis for children with ALL has substantially improved with the use of multi-agent therapy over the last few decades. Unfortunately, two opposing challenges remain in childhood ALL treatment: relapse and toxicity. One of toxicities from ALL therapy consists in avascular necrosis (AVN). While the cause of AVN is multi-factorial, significant high-risk factors include high cholesterol and treatment with corticosteroids. Statins, such as Simvastatin, widely used as a cholesterol-lowering drug, have demonstrated cytostatic properties against several types of cancer cell lines. In previous studies, our group has confirmed some the anticancer and anti-CSC effects in breast cancer, both in vivo and in vivo. In this context, we evaluated for the first time the cytotoxic, anti-cancer stem cells and anti-proliferative effects of Simvastatin, either isolated or combined with daunorubicin on MOLT-4 and REH cell lines which are in vitro models for human ALL.

OBJETIVOS: Acute lymphoblastic leukemias (ALL) are malignant disorders of immature B and T cells that occur characteristically in children, generally under the age of 6, representing 80% of childhood leukemias. Although therapeutic regimens for the pediatric age group have evolved in recent decades, in adulthood, the cure rate still remains close to 40%. The prognosis for children with ALL has substantially improved with the use of multi-agent therapy over the last few decades. Unfortunately, two opposing challenges remain in childhood ALL treatment: relapse and toxicity. One of toxicities from ALL therapy consists in avascular necrosis (AVN). A recent research suggested over 70% of children with this cancer develop AVN. While the cause of AVN is multi-factorial, significant high-risk factors include high cholesterol and treatment with corticosteroids. Statins, such as Simvastatin, widely used as a cholesterol-lowering drug, have demonstrated cytostatic properties against several types of cancer cell lines. In previous studies, our group has confirmed some the anticancer and anti-CSC effects in breast cancer, both in vivo and in vivo. In this context, we evaluated for the first time the cytotoxic, anti-cancer stem cells and anti-proliferative effects of Simvastatin, either isolated or combined with daunorubicin on MOLT-4 and REH cell lines which are in vitro models for human ALL.

MÉTODOS: MOLT-4 T Acute Lymphoblastic Leukemia and REH non-T/non-B Acute lymphocytic leukemia cell lines (5x104/well/each cell) were exposed to simvastatin (SNV), daunorubicin (DN) or a combination of both drugs, in 96-well microtiter plate, for 72 hours. At the end of treatment protocols, the cells were assessed for viability using MTT assays. The absorbance was read in a SpectraMax 340PC 384 microplate reader (Molecular Device, 1311 Orleans Drive Sunnyvale, CA 94089) at 570 nm. The results were expressed as IC50 and compared to those of the control drug (daunorubicin). IC50 values were reported as mean ± standard deviation (SD) of two independent experiments, each performed in quintuplicate. Then, each cell line was treated with IC50 values of each drug alone, or the combination with IC25 values (IC25Daunorubicin+IC25Simvastatin). The effect of these drugs on protein expression of Ki-67 (cell proliferation marker) and stem cell marker CD34 was determined by immunocytochemistry. One-way analysis of variance (ANOVA) was used for comparing groups and the differences were assessed by a Tukey’s post hoc test.

RESULTADOS: The IC50 values of simvastatin (SNV) and daunorubicin (DN) were, respectively: 0.308±0.390 and 0.002±0.001 for MOLT-4 cells; 0.735±0.517 and 0.002±0.002 for REHs, at 72 h. These results demonstrate that, though less potent than the control drug (daunorubicin), simvastatin is significantly cytotoxic at low doses, on both types of ALL cell lines (thus confirming its antineoplastic effect). Both leukemic cell lines were characterized by a monotonous proliferation of round lymphoid cells, with hyperchromatic nuclei and scarce amphophilic cytoplasm. None of these morphological features were significantly altered by pharmacological treatments, regardless of cell line type. Immunostaining for CD34 and Ki67 were significantly reduced in REH and MOLT4 cells by all tested treatments (with no significant differences between treatments) (p<0.05).

CONCLUSÃO: The IC50 values of simvastatin (SNV) and daunorubicin (DN) were, respectively: 0.308±0.390 and 0.002±0.001 for MOLT-4 cells; 0.735±0.517 and 0.002±0.002 for REHs, at 72 h. These results demonstrate that, though less potent than the control drug (daunorubicin), simvastatin is significantly cytotoxic at low doses, on both types of ALL cell lines (thus confirming its antineoplastic effect). Both leukemic cell lines were characterized by a monotonous proliferation of round lymphoid cells, with hyperchromatic nuclei and scarce amphophilic cytoplasm. None of these morphological features were significantly altered by pharmacological treatments, regardless of cell line type. Immunostaining for CD34 and Ki67 were significantly reduced in REH and MOLT4 cells by all tested treatments (with no significant differences between treatments) (p<0.05).

BIBLIOGRAFIA: SWERDLOW SH, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4 ed. Lyon, 2017

PALAVRA-CHAVE: Acute lymphocytic leukemia, Simvastatin, Daunorubicin, anti-neoplastic drugs.