The Potent Humanin Analogue (HNG) Protects Germ Cells and Leucocytes While Enhancing Chemotherapy-Induced Suppression of Cancer Metastases in Male Mice

Abstract

Humanin is a peptide that is cytoprotective against stresses in many cell types. We investigated whether a potent humanin analogue S14G-humanin (HNG) would protect against chemotherapy-induced damage to normal cells without interfering with the chemotherapy-induced suppression of cancer cells. Young adult male mice were inoculated iv with murine melanoma cells. After 1 week, cancer-bearing mice were randomized to receive either: no treatment, daily ip injection of HNG, a single ip injection of cyclophosphamide (CP), or CP+HNG and killed at the end of 3 weeks. HNG rescued the CP-induced suppression of leucocytes and protected germ cell from CP-induced apoptosis. Lung metastases were suppressed by HNG or CP alone, and further suppressed by CP+HNG treatment. Plasma IGF-1 levels were suppressed by HNG with or without CP treatment. To investigate whether HNG maintains its protective effects on spermatogonial stem cells, sperm output, and peripheral leucocytes after repeated doses of CP, normal adult male mice received: no treatment, daily sc injection of HNG, 6 ip injections of CP at 5-day intervals, and the same regimens of CP+HNG and killed at the end of 4 weeks of treatment. Cauda epididymal sperm counts were elevated by HNG and suppressed by CP. HNG rescued the CP-induced suppression of spermatogonial stem cells, sperm count and peripheral leucocytes. We conclude that HNG 1) protects CP-induced loss of male germ cells and leucocytes, 2) enhances CP-induced suppression of cancer metastases, and 3) acts as a caloric-restriction mimetic by suppressing IGF-1 levels. Our findings suggest that humanin analogues may be promising adjuvants to chemotherapy.

Figure 1.

Representative photographs of glutaraldehyde-fixed lungs with metastatic melanomas from the NT, HNG-treated alone (HNG), CP alone (CP), and combined CP with HNG-treated (CP+HNG) mice inoculated with 2 × 10⁵ B16 cells per mouse in experiment 1 (n = 10 mice per group) (A). The bar graph shows the number (B) and the size (C) of lung metastatic tumors. Values are the mean ± SEM. CP = cyclophosphamide; HNG = S14G-humanin

Figure 2.

Plasma humanin/HNG (A), IGF-1 (B), and IGFBP-1 (C) levels in the NT, HNG-treated alone (HNG), CP alone (CP), and combined CP with HNG-treated (CP+HNG) mice in experiment 2 (n = 5 mice per group). Values are mean ± SEM.

Figure 3.

Peripheral blood cell counts of WBC (A), granulocytes (B), monocytes (C), and lymphocytes (D) in the NT, HNG-treated alone (HNG), CP alone (CP), and combined CP with HNG-treated (CP+HNG) mice in experiment 2 (n = 5 mice per group). Values are mean ± SEM.

Figure 4.

Representative immunohistochemistry micrographs of mouse testis sections from control (n = 5) (A), HNG daily sc injection (n = 5) (B), 6 repeated dose of CP treatment (n = 10) (C), and CP+HNG-treated mice (n = 10) (D); scale bar, 0.05 mm. The cross-sections of seminiferous tubules containing elongated spermatids (red arrows) were noted both in CP (C) and CP+HNG (D)-treated mice. The FoxO1-positive SSCs (dark brown in color, black arrows) were observed in testes from all groups. The bar graph (E) shows the percentage of seminiferous tubules containing germ cells in control, HNG-treated alone, repeated CP alone, and combined CP with HNG-treated (CP+HNG, n = 10) mice. The bar graph (F) shows the number of SSCs (FoxO1-positive spermatogonia) per 100 cross-sections of seminiferous tubules from control (Con), HNG-treated alone, repeated CP alone, and combined CP with HNG-treated (CP+HNG) mice. Values are mean ± SEM.

Figure 5.

Cauda epididymal sperm count (million/cauda) in control (Con) (n = 5), HNG-treated alone (HNG) (n = 5), CP alone (CP) (n = 10), and combined CP with HNG-treated (CP+HNG) (n = 10) mice. Values are mean ± SEM.