Clin Exp Otorhinolaryngol.  2010 Dec;3(4):217-225. 10.3342/ceo.2010.3.4.217.

Growth Inhibition and Apoptosis with H31 Metabolites from Marine Bacillus SW31 in Head and Neck Cancer Cells

• Lim YC
• Cho KW
• Kwon HC
• Kang SU
• Pyun JH
• Lee MH
• Hwang HS
• Kim JH
• Lee HN
• Choi EC
• Kim CH

• KMID: 2049055
• DOI: http://doi.org/10.3342/ceo.2010.3.4.217

Abstract

OBJECTIVES
To determine whether a novel marine micro-organism with anticancer properties, H31, the metabolic product of Bacillus SW31, has anti-tumor effects on head and neck cancer, and potential for apoptotic-enhancing anti-cancer treatment of affected patients.
METHODS
The cell viability and apoptosis assays were performed. Changes in the signal pathway related to apoptosis were investigated. Then, the therapeutic effects of H31 were explored in mouse xenograft model and drug toxicity of H31 was examined in zebrafish model.
RESULTS
We identified the anticancer activity of H31, a novel metabolic product of Bacillus SW31. Bacillus SW31, a new marine micro-organism, has 70% homology with Bacillus firmus and contains potent cytotoxic bioactivity in head and neck cancer cells using MTT assay. Combined with c-JUN, p53, cytochrome C, and caspase-3, H31 induced apoptosis of KB cells, a head and neck cancer cell line. In a separate in vivo model, tumor growth in C3H/HeJ syngeneic mice was suppressed by H31. In addition, in a zebrafish model used for toxicity testing, a considerable dose of H31 did not result in embryo or neurotoxicity.
CONCLUSION
Growth inhibition and apoptosis were achieved both in vitro and in vivo in head and neck cancer cells after exposure to H31, a metabolite from the marine Bacillus species, without any significant toxicity effects even at considerable H31 dose concentrations.

Keyword

Apoptosis; Bacillus; Marine toxins; Head and neck cancer; Cytotoxicity

MeSH Terms

Animals
Apoptosis
Bacillus
Caspase 3
Cell Line
Cell Survival
Cytochromes c
Drug Toxicity
Embryonic Structures
Head
Head and Neck Neoplasms
Humans
KB Cells
Marine Toxins
Mice
Signal Transduction
Toxicity Tests
Transplantation, Heterologous
Zebrafish
Caspase 3
Cytochromes c
Marine Toxins

Figure

• 

  Fig. 1 Four head and neck cancer cell lines (FaDu, SNU 899, SNU 1066, and KB) were incubated with different concentrations (1, 10, 100 µg/mL) of H31 for 24 hours, and cell viability was evaluated by the MTT assay. The data represent the mean±SD of three independent experiments; **P<0.01, ***P<0.001 by student t-test, compared to medium alone.

• 

  Fig. 2 Effect of H31 on apoptosis and the cell cycle in KB cells. (A) DNA fragmentation analysis: KB cells were grown for 24 hours at different concentrations of H31; a DNA fragmentation assay was performed with an ApopLadder EX DNA fragmentation assay kit. DNA fragmentation was visualized by electrophoresis on a 2% agarose gel containing ethidium bromide. lane 1, control; lane 2, H31 30 µg/mL; lane 3, H31 50 µg/mL; lane 4, H31 70 µg/mL; lane 5, H31 100 µg/mL. (B) TUNEL study: apoptosis in KB cells was determined by the TUNEL method using an in situ cell detection kit. The cells were then incubated with 50 µL of TUNEL reaction mixture (TdT and fluoresccin-dUTP) and stained with Hoechst 33258 (5 µg/mL). The stained cells (arrowhead) were observed under a fluorescence microscope. Scale bar=50 µm. (C) Flow cytometry: KB cells were incubated in 6-well plates with various concentrations of H31, treated with 200 mg/mL RNase A, and stained with 5 mg/mL PI. The stained cells were then analyzed with a FACS caliber instrument. (D) Annexin V and PI staining. To quantify the effects of H31 on apoptosis, annexin V-FITC and propidium iodide staining were used to analyze the percentage of apoptotic cells treated with H31. The data represent the mean±SD of three independent experiments. **P<0.01, ***P<0.001 by student t-test, compared to medium alone.

• 

  Fig. 3 Expression of apoptosis-associated proteins: KB cells were treated with H31 at the indicated concentrations. Serum-starved KB cells were treated with different concentrations of H31, and then Western blotting was performed with various antibodies, including p-JNK, p-p53, cytochrome C, procaspase 3, and cleaved caspase-3.

• 

  Fig. 4 Effect of H31 on tumor growth and apoptosis in C3H/HeJ syngeneic mice. Six C3H/HeJ syngeneic mice with subcutaneous injections of 5×105 SCC VII cells were divided into two groups. Three were then given daily intraperitoneal injections of distilled water (control) and the other 3 mice were treated with 0.2 mg H31. After 22 days, the mice were sacrificed and tumors were removed. (A) Effect of H31 on tumor volume. (B) Effect of H31 on tumor weight. (C) Effect of H31 on tumor growth (time course). (D) Gross appearance. (E) Tissue TUNEL staining. (F) Immunohistochemistry for caspase-3. *P<0.01, **P<0.05.

• 

  Fig. 5 Zebrafish assay for the evaluation of embryotoxicity and neurotoxicity of H31: (A) Embryotoxicity: wild type zebrafish were maintained at 28.5℃ on a 14 hours light/10 hours dark cycle, then fertilized eggs were exposed to the indicated concentrations of H31 or 100 µM cisplatin, and maintained in a incubator at 28.5℃ for 1 hour. After H31 treatment, egg death, organogenesis, hatch rate, and any abnormal features were evaluated; (B) Neurotoxicity: The 4 dpf zebrafish were treated with cisplatin (50 µM, 100 µM) or increasing concentrations of H31 for 24 hours and then were stained with YO-PRO1. White dots on the body of zebrafish indicate neuromasts.

• 

  Fig. 6 Extraction of a pure cytotoxic compound (PCC) of H31 using the HPLC method and results of the MTT assay: The PCC showed the highest peak (black arrow) and the HPLC showed strong cytotoxicity to KB cells in the concentration of 10 µg/mL. This single compound appears to possess the anti-tumor capabilities of H31. ***P<0.001.

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