The humanin analogue (HNG) prevents temozolomide-induced male germ cell apoptosis and other adverse effects in severe combined immuno-deficiency (SCID) mice bearing human medulloblastoma

Abstract

Subfertility is a major concern of long-term cancer survivors at the reproductive age. We have previously demonstrated that a potent humanin analogue, HNG, protected chemotherapy-induced apoptosis in germ cells but not cancer cells in a metastatic melanoma allograft model. In this study, we utilized severe combined immuno-deficiency (SCID) mice bearing human medulloblastoma to study the effect of HNG in Temozolomide (TMZ) induced male germ cell apoptosis and white blood cell (WBC) suppression. Human medulloblastoma DAOY cells were injected subcutaneously into the right flank of male SCID mice. Three weeks later, groups of tumor-bearing mice received one of the following treatments: vehicle, HNG, TMZ, or TMZ + HNG. 24 h after last injection, the tumors weights, complete blood counts, liver and spleen weights, male germ cell apoptosis was assessed. HNG did not affect TMZ's significant anti-tumor action. HNG significantly prevented TMZ-induced germ cell apoptosis and attenuated the suppressed total WBC and granulocyte counts in SCID mice with or without TMZ treatment. HNG also attenuated TMZ-induced body weight loss and decrease of spleen and liver weights. In conclusion, HNG ameliorated TMZ-induced germ cell apoptosis; WBC and granulocytes loss; and decreased body/organ weights without compromising the TMZ's anti-cancer action on medulloblastoma xenografts in SCID mice.

Keywords: Adverse effect; Humanin analogue; Medulloblastoma; SCID mice; Temozolomide.

FIG.1.. Effect of HNG on Temozolomide (TMZ)-induced weight changes of body, spleen, and liver

Mice were treated with vehicle (Control), HNG, TMZ, TMZ+HNG as described in experimental procedures (n=4 in control, 5 each group in the rest). (A) HNG had no significant effect while TMZ significantly decreased body weights when compared to control. HNG significantly prevented TMZ-induced body weights loss (P<0.05). (B) HNG alone increased spleen weights compared with control SCID mice. TMZ significantly decreased spleen weight which were prevented by combined HNG administration (P<0.05). (C) HNG alone did not change liver weight while TMZ significantly decreased liver weight. HNG partially prevented TMZ-suppressed liver weights (P<0.05). Values are means ± SEM.

FIG.2.. Effects of HNG on Temozolomide (TMZ)-induced changes of cleaved PARP and phosphorylated STAT3 in liver and spleen

Protein expressions in liver and spleen were measured by Western blots and optical density ratio was used to compare protein expression changes between different groups. (A) Changes of cleaved PARP in liver tissue. Compared with control, HNG alone did not change cleaved PARP. HNG significantly attenuated TMZ-increased cleaved PARP. Uncleaved PARP was used as nomalizer. (B) Changes of phosphorylated STAT3 in liver tissue. Compared with control, HNG alone did not change phosphorylation of STAT3. HNG significantly attenuated TMZ-suppressed STAT3 phosphorylation. STAT3 was not different in any treatment group. (C) Changes of cleaved PARP in spleen tissue. Compared with control, HNG suppressed cleaved PARP in SCID mice with or without TMZ treatment. Uncleaved PARP was used as nomalizer. (D) Changes of phosphorylated STAT3 in spleen tissue. Compared with control, HNG increased phosphorylation of STAT3 in SCID mice with or without TMZ treatment. STAT3 was not different in any treatment group. Values are means ± SEM.

FIG.3.. Effect of HNG on Temozolomide (TMZ)-induced male germ cell apoptosis in SCID mice

Quantification of apoptotic germ cells in early+late stages (A) and middle stages (B) in SCID mice testis. The rate of germ cell apoptosis is similar between control and HNG groups (p>0.05). TMZ significantly increased the number of apoptotic cells compared with control (p<0.05). HNG co-treatment significantly attenuated germ cell apoptosis induced by TMZ treatment (p<0.05). Values are the mean ± SEM.

FIG.4.. Effect of HNG on Temozolomide (TMZ)-induced changes of STAT3, p38MAPK, BAX, and Cytochrome C in testis

Protein expressions in testis were measured by Western blots and optical density ratio was used to compare protein expression changes between different groups. (A) Compared with control, HNG alone did not change phosphorylation of STAT3. HNG significantly attenuated TMZ-suppressed STAT3 phosphorylation. STAT3 was not different in any treatment group. (B) Change of p38MAPK phosphorylation were detected by an activity kit and presented by optical density ratio of phosphorylated ATF2/ATF2 as described in methods. Compared with control, HNG alone did not change phosphorylation of p38MAPK. HNG co-treatment significantly suppressed TMZ-induced p38MAPK activation. (C) Compared with control, HNG alone did not affect BAX expression in the mitochondria (COX IV as a loading control for mitochondrial fraction). HNG co-treatment prevented TMZ-induced BAX increase in the mitochondria. (D) Compared with control, HNG alone did not change cytochrome c expression in the cytoplasmic fraction. HNG co-treatment partially prevented TMZ-induced increased in cytochrome c in the cytoplasm. Values are means ± SEM.

FIG.5.. Effect of HNG on white blood cell and differential counts in SCID mice

Mice were treated with vehicle (Control), HNG, TMZ, TMZ+HNG as described in experimental procedures (n=3 in control, 4 in TMZ group, and 5 each group in the rest). HNG alone increased total WBC (A) and granulocytes (B) compared with non-treated control SCID mice. Compared with control, TMZ did not suppress WBC but TMZ+HNG significantly increased WBC (A) and granulocytes number (B). No significant change was detected in lymphocytes (C) and monocytes (D) number between different groups. Values are means ± SEM.

FIG.6.. HNG did not change anti-tumor effects of Temozolomide (TMZ)

Mice were treated with vehicle (Control), HNG, TMZ, TMZ+HNG as described in experimental procedures (n=4 in control, 5 each group in the rest). (A) Before treatment, there was no difference of tumor sizes between treatment groups. After treatment, tumor growth was measured by tumor size (B) and tumor weight (C). HNG alone did not change tumor size or weight compared with control SCID mice. TMZ significantly suppressed tumor growth. HNG-TMZ co-treatment did not further enhance TMZ-suppressed tumor growth. Values are means ± SEM.