A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer.

Methods:

Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated.

Results:

A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed.

Conclusion:

Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.

Keywords:
Bone metastasis; Melittin; Intratumoral injection; Colorectal neoplasms; Bee venom

Authorship

Mackson Martins Rocha

Multidisciplinary Laboratory, Postgraduate Program in Health Sciences, São Francisco University, Bragança Paulista, SP, Brazil. São Francisco UniversityBrazilBragança Paulista, SP, BrazilMultidisciplinary Laboratory, Postgraduate Program in Health Sciences, São Francisco University, Bragança Paulista, SP, Brazil.

& These authors contributed equally to this work and share the first authorship

Isabela Dariva

Medical School, São Francisco University, Bragança Paulista, SP, Brazil. São Francisco UniversityBrazilBragança Paulista, SP, BrazilMedical School, São Francisco University, Bragança Paulista, SP, Brazil.

& These authors contributed equally to this work and share the first authorship

Gabriela Comelli Zornoff

& These authors contributed equally to this work and share the first authorship

Giovanna Sanches De Laurentis

Giulia Carli Mendes

Maycon Giovani Santana

Nursing School, São Francisco University, Bragança Paulista, SP, Brazil.São Francisco UniversityBrazilBragança Paulista, SP, BrazilNursing School, São Francisco University, Bragança Paulista, SP, Brazil.

http://orcid.org/0000-0003-2084-668X

Guilherme Chohfi de Miguel

Rui Seabra Ferreira Junior

Center for the Studies of Venoms and Venomous Animals (CEVAP), São Paulo State University (UNESP), Botucatu, SP, Brazil.São Paulo State University (UNESP)BrazilBotucatu, SP, BrazilCenter for the Studies of Venoms and Venomous Animals (CEVAP), São Paulo State University (UNESP), Botucatu, SP, Brazil.

http://orcid.org/0000-0001-6952-0512

Juliana Mozer Sciani

http://orcid.org/0000-0001-7213-206X

Denise Gonçalves Priolli ^**Correspondence:depriolli@terra.com.br; denise.priolli@usf.edu.br.

http://orcid.org/0000-0003-3190-4013

*Correspondence:depriolli@terra.com.br; denise.priolli@usf.edu.br.

Competing interests

RSF Jr is the editor-in-chief of Journal of Venomous Animals and Toxins including Tropical Diseases. He did not get involved in the peer review process of this manuscript.

Authors’ contributions

MMR, ID, GCZ, GCM, MGS and RSF Jr. were responsible for the initial research. MMR, ID, GCZ and GCM drafted the work. JMS and DGP designed the study, analyzed the results, carried out data interpretation, as well as drafted the work or substantively revised it. Project administration and funding acquisition was conducted by DPG. All authors read and approved the final manuscript.

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Figures

Figures (5)

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Figure 1.
Characterization of melittin used in the CRC treatment. (A) Mass spectrometry analysis showing purity and molecular mass of the peptide. (B) MTT assay with antiproliferative results of melittin on HT-29/carcinoma colon in vitro (mean ± SD). The dose-response relationship demonstrates the effect with increasing dose levels (p < 0.05), which reached its maximum with 2 µM resulting in 80% inhibition.

Thumbnail

Figure 2.
Timeline and CRC bone metastasis progression. (A) On day 19: puncture site by melittin intratumoral injection. (B) On day 20: inflammatory response one day after melittin. (C) On day 27: evident reduction of metastasis size.

Thumbnail

Figure 3.
In vivo efficacy of melittin. CRC bone metastasis (A, B) one day before melittin intrametastatic injection and (C, D) seven days after a single dose of melittin (day 27). (E) Metastasis normalized volume (mean ± SD) evolution in untreated (n = 3) or melittin-treated animals (n = 6) (p < 0.001). (C, D) After intrametastatic melittin injection, bone metastases are smaller than in (A, B) untreated animals. Seven days after melittin injection, metastases started growing again.

Thumbnail

Figure 4.
Histopathological characteristics of bone metastasis in colon cancer following inoculation of xenogeneic HT-29 tumor cells (A, B, C) in untreated or (D, E, F) melittin-treated mice (day 27). (A B, C) Metastasis-bearing untreated mice exhibit intense cancer cell invasion into intertrabecular spaces (ITS) characterized by lytic metastasis with bone infiltration, presence of glandular tissue (GT and arrows) and solid sheet areas. (D, E, F) Metastasis-bearing melittin-treated mice exhibit inflammatory infiltrated (II) and necrosis (N) areas without cancer cells. Untreated animals (n = 3); melittin-treated animals (n = 6). H&E, A [4040. Mahmoodzadeh A, Zarrinnahad H, Bagheri KP, Moradia A, Shahbazzadeh D. First report on the isolation of melittin from iranian honey bee venom and evaluation of its toxicity on gastric cancer AGS cells. J Chin Med Assoc. 2015 Oct;78(10):574-83. doi: 10.1016/j.jcma.2015.06.008.
https://doi.org/10.1016/j.jcma.2015.06.0... ×], B, D and E [100×], C and F [400×].

Thumbnail

Figure 5.
Body weight and histological structure of leaver, kidney and heart following seven days after a single dose of melittin treatment (day 27) showing absence of structural toxicity of melittin. (A) Graph showing normalized animal body weight (mean ± SD) over time after the day of melittin injection (day 19) in untreated or melittin-treated animals; weight loss is observed in animals treated with melittin (p < 0.01). Note the stabilization of body weight 24 h after melittin injection. (B) Liver micrograph of melittin group showing normal histological structure of the central vein (CV) and surrounding hepatocytes (H) and sinusoids. (C) Kidney micrograph of melittin group showing normal histological structure of the glomerulus (G), Bowman’s capsule (BC), urinary polo (UP) and distal convoluted tubule (DCT). (D) Cardiac muscle micrograph showing normal histological structure of the striated cardiac muscle, single central nucleus (MN) for each cell and connective tissue (CT) between muscle cells. Untreated animals (n = 3); melittin-treated animals (n = 6). H&E, B [200×], C and D [100×].

Figure 1. Characterization of melittin used in the CRC treatment. (A) Mass spectrometry analysis showing purity and molecular mass of the peptide. (B) MTT assay with antiproliferative results of melittin on HT-29/carcinoma colon in vitro (mean ± SD). The dose-response relationship demonstrates the effect with increasing dose levels (p < 0.05), which reached its maximum with 2 µM resulting in 80% inhibition.

Figure 2. Timeline and CRC bone metastasis progression. (A) On day 19: puncture site by melittin intratumoral injection. (B) On day 20: inflammatory response one day after melittin. (C) On day 27: evident reduction of metastasis size.

Figure 3. In vivo efficacy of melittin. CRC bone metastasis (A, B) one day before melittin intrametastatic injection and (C, D) seven days after a single dose of melittin (day 27). (E) Metastasis normalized volume (mean ± SD) evolution in untreated (n = 3) or melittin-treated animals (n = 6) (p < 0.001). (C, D) After intrametastatic melittin injection, bone metastases are smaller than in (A, B) untreated animals. Seven days after melittin injection, metastases started growing again.

Figure 4. Histopathological characteristics of bone metastasis in colon cancer following inoculation of xenogeneic HT-29 tumor cells (A, B, C) in untreated or (D, E, F) melittin-treated mice (day 27). (A B, C) Metastasis-bearing untreated mice exhibit intense cancer cell invasion into intertrabecular spaces (ITS) characterized by lytic metastasis with bone infiltration, presence of glandular tissue (GT and arrows) and solid sheet areas. (D, E, F) Metastasis-bearing melittin-treated mice exhibit inflammatory infiltrated (II) and necrosis (N) areas without cancer cells. Untreated animals (n = 3); melittin-treated animals (n = 6). H&E, A [4040. Mahmoodzadeh A, Zarrinnahad H, Bagheri KP, Moradia A, Shahbazzadeh D. First report on the isolation of melittin from iranian honey bee venom and evaluation of its toxicity on gastric cancer AGS cells. J Chin Med Assoc. 2015 Oct;78(10):574-83. doi: 10.1016/j.jcma.2015.06.008.
https://doi.org/10.1016/j.jcma.2015.06.0... ×], B, D and E [100×], C and F [400×].

Figure 5. Body weight and histological structure of leaver, kidney and heart following seven days after a single dose of melittin treatment (day 27) showing absence of structural toxicity of melittin. (A) Graph showing normalized animal body weight (mean ± SD) over time after the day of melittin injection (day 19) in untreated or melittin-treated animals; weight loss is observed in animals treated with melittin (p < 0.01). Note the stabilization of body weight 24 h after melittin injection. (B) Liver micrograph of melittin group showing normal histological structure of the central vein (CV) and surrounding hepatocytes (H) and sinusoids. (C) Kidney micrograph of melittin group showing normal histological structure of the glomerulus (G), Bowman’s capsule (BC), urinary polo (UP) and distal convoluted tubule (DCT). (D) Cardiac muscle micrograph showing normal histological structure of the striated cardiac muscle, single central nucleus (MN) for each cell and connective tissue (CT) between muscle cells. Untreated animals (n = 3); melittin-treated animals (n = 6). H&E, B [200×], C and D [100×].

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A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

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[1] *Correspondence:depriolli@terra.com.br; denise.priolli@usf.edu.br.