Targeting Sphingosine Kinase 1 and Apoptosis by Metformin

Targeting Sphingosine Kinase 1 and Apoptosis by Metformin to Decrease Tumor Resistance to Adriamycin By Dr. Ahmed Mohamed Kabel Pharmacology Department, Faculty of Medicine, Tanta University, Egypt Pharmacology Department, College of Pharmacy, Taif University, KSA

• Adriamycin (ADR, Doxorubicin) is an anthracycline antibiotic that is frequently used as a treatment for many types of cancer such as leukemia, lymphoma, breast, ovarian and lung cancer. • It damages DNA by intercalation into DNA and inhibition of topoisomerase II resulting in DNA strand breaks.

• The major limiting factor for the use of ADR in cancer therapy is the development of resistance. • The mechanisms of this resistance may include increased activity of sphingosine kinase-1 (Sph. K 1) enzyme which inhibits penetration and intercalation of ADR into DNA and significantly abolishing the anticancer effect of ADR. • This makes it essential to combine ADR with agents that inhibit these mechanisms to decrease the incidence of resistance of cancer cells to ADR.

• Metformin is an antidiabetic agent that decreases intestinal absorption of glucose, increases its anaerobic metabolism and improves insulin sensitivity. • Studies on animal models had demonstrated that metformin prevents tumor development and inhibits cell proliferation. This effect may be mediated through its regulatory role on the hormonal, metabolic and immune functions.

• The main molecular target of metformin is AMPactivated protein kinase (AMPK) signaling that plays a crucial role in the control of cell division and proliferation. • Moreover, metformin had been shown to improve endothelial function, decrease inflammatory response, and regulate immune functions which have a major role in the pathogenesis of cancer. • Also, metformin was proven to inhibit Sph. K 1 activity which was one of the main factors contributing to resistance of cancer cells to chemotherapy.

So, The aim of this work was to study the effect of targeting sphingosine kinase 1 and apoptosis by metformin on tumor resistance to adriamycin using transplantable tumor model in mice. a

• In this study, we used a model of solid Ehrlich carcinoma (SEC), where Ehrlich carcinoma cells (ECCs) were implanted subcutaneously into the right thigh of the hind limb of mice. A palpable solid tumor mass (about 100 mm 3) was developed within 10 days.

Ehrlich Ascites Carcinoma Used for induction of SEC

SEC Control

One hundred BALB/C mice were divided into 5 equal groups of twenty mice each as follows: Group (1): the Control untreated group Group (2): ECCs were implanted subcutaneously into the right thigh of the hind limb of mice. Group (3): Adriamycin was given by intra-tumoral injection on days 10, 15, 20, 25, 30 and 35 after implantation of ECCs.

Group (4): Metformin was given to mice orally starting 10 days after implantation of ECCs and continued for 32 days. Group (5): Adriamycin and metformin were given together starting 10 days after implantation of ECCs and continued for 32 days by the above regimens.

• Tumor volume was measured on days 15, 20, 25, 30, 35 and 40 after implantation of ECCs. • In the 42 nd day after implantation of ECCs, the animals were killed and the tumor was excised and divided into two portions: one for homogenization and the other for histopathological examination. • The tumor tissue was homogenized for determination of tissue catalase, glutathione reductase, malondialdehyde, sphingosine kinase 1 activity, caspase 3 activity and tumor necrosis factor alpha.

Effect of different treatments on tumor volume a Significant compared to SEC group (P < 0. 05). b Significant compared to SEC+ADR group (P < 0. 05). c Significant compared to SEC+Metformin group (P < 0. 05).

300 250 30 Tissue MDA umol/gm Tissue catalase U/mg tissue 35 25 20 15 10 5 200 150 100 50 0 0 Control SEC ADR+SEC Met+SEC Control ADR+Met+SEC ADR+SEC Met+SEC ADR+Met+SEC Tissue glutathione reductase U/g 1000 900 800 700 600 500 400 300 200 100 0 Control SEC ADR+SEC Met+SEC ADR+Met+SEC Effect of different treatments on tissue antioxidant parameters

1600 Tissue TNF alpha pg/g 1400 1200 1000 800 600 400 200 0 Control SEC ADR+SEC Met+SEC ADR+Met+SEC Effect of different treatments on tissue TNF-α

Tissue caspase 3 activity nmol/mg protein/min 10 9 8 7 6 5 4 3 2 1 0 Control SEC ADR+SEC Met+SEC ADR+Met+SEC Effect of different treatments on tissue caspase 3 activity

Tissue Sph. K 1 activity pmol/min/mg protein 70 60 50 40 30 20 10 0 Control SEC ADR+SEC Met+SEC ADR+Met+SEC Effect of different treatments on tissue Sph. K 1 activity

Apoptotic index % 7 6 5 4 3 2 1 0 Control SEC ADR+SEC Met+SEC ADR+Met+SEC Effect of different treatments on the apoptotic index

H&E stained sections of a) SEC showing sheets of small tumor cells representing cell proliferation surrounding areas of necrosis; b) ADR group showing focal necrosis with sheets of malignant cells; c) Metformin showing focal necrosis; d) ADR/metformin combination group showing extensive necrosis.

A photomicrograph of a) SEC group showing negative staining for p 53; b) ADR group showing positive (+++) p 53 expression; c) Metformin group showing positive (++) p 53 expression; d) ADR/metformin group showing positive (++++) p 53 expression.

Conclusions • The combination of ADR and metformin had a better effect than each of these drugs alone against transplantable tumor model in mice. • This might be due to the combined antioxidant and anti-inflammatory properties of both drugs together with their ability to induce apoptosis of cancer cells. • Moreover, metformin was able to decrease Sph. K 1 enzyme activity which potentiates the effect and decreases resistance of cancer cells to ADR.

• So, it is recommended to add metformin to the anti-cancer regimens containing ADR to decrease resistance of cancer cells to ADR.