Introduction
Canstatin and Tumstatin are endogenous angiogenesis inhibitor proteins derived from the α2 and α3 chains of collagen IV, respectively. They significantly prevent angiogenesis and tumor growth in vitro and animal models [13]. Two overlapping peptides from Tumstatin, T3 (amino acids 69–88) and T7 (amino acids 74–98), are more potent than other fragments [14]. Since short peptides may have advantages over longer ones in certain aspects, such as more convenient synthesis, we recently compared the antiangiogenic and antitumor activities of a 9-amino acid peptide derived from Tumstatin (amino acids 78-86) with its corresponding sequence in Canstatin. The results showed that both peptides significantly inhibited proliferation, migration, and vascular tube formation in endothelial cells, as well as colon tumor growth in mice. 
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is overexpressed and activated in several solid cancers, promoting tumor progression and metastasis. FAK controls cell motility, invasion, survival, and self-renewal of cancer stem cells [18]. FAK messenger ribonucleic acid (mRNA) levels are increased in several tumor types and inversely correlated with overall patient survival. Given the importance of FAK in the growth of cancer cells and clarification of the antitumor mechanism of peptides derived from Tumastatin (peptide T) and Canstatin (peptide C), this study aimed to investigate the effect of these peptides on breast tumor growth in mice and the expression of FAK in tumor tissue. No study has investigated the effect of peptides derived from these proteins on FAK expression. The present study sheds light on another aspect of their mechanism of action.

Methods 
Peptides were synthesized by Shine Gene Biotechnology Inc. (Shanghai, China) with the amino acid sequence +3HN-LYCNPGDVC-COO- for peptide C and +3HN-LFCNVNDVC-COO- for peptide T.
A suspension containing 5×105 4T1 cells (a metastatic breast cell line) was injected s.c. into the right flank of mice. When the tumor volume reached approximately 200 mm3, tumor-bearing mice were randomly divided into control and treatment groups (n=3) and received phosphate-buffered saline (PBS) and 5 mg/kg/day of peptides (i.p. injection), respectively, for twelve days. Tumor dimensions were measured, and tumor volumes were calculated using the Equation 1:

1. v= a²×b×0.52, 

where a is the smallest and b is the largest tumor diameter. Data were represented as Mean±SE. 
RNA was extracted from tumor tissues according to the manufacturer’s protocol. The primers required for the amplification of FAK and GAPDH genes (Table 1) were designed using this software. The Pars Toos kit was used for Complementary DNA (cDNA) synthesis. For real-time polymerase chain reaction (PCR), appropriate volumes of Mastermix, cDNA, and primers were mixed and brought up to 20 μL with sterile double-distilled water. The four-step PCR was programmed as follows: A 15-minute holding step at 95 °C, an initial 30-second denaturation step at 95 °C, a 30-second annealing step at 65 °C, and a 30-second extension step at 72 °C. All programs were run with at least three independent cDNA samples. Finally, the cycle threshold (CT) values of genes in the treated and control samples were calculated. The CT of the FAK was subtracted from the CT of the GAPDH, and the ΔCT value was obtained for the treated and control samples. The relative expression graph was displayed based on 2-ΔCT. Data were represented as Mean±SE. Statistical significance was set at P<0.05.



Results 
Peptides C and T prevented tumor growth by 51% and 49%, respectively, compared to the control group (Figure 1) (P<0.05).



However, the difference between the two treatment groups was not significant. Peptides C and T reduced FAK expression by 69% and 67%, respectively, compared to the control group (P<0.05) (Figure 2). No significant difference was observed between the effects of the two peptides on FAK expression.



Conclusion 
The present study showed that short antiangiogenic peptides derived from collagen IV can inhibit breast tumor growth in mice by approximately 50% and reduce the expression of FAK, a crucial gene involved in the growth, proliferation, and metastasis of cancer cells. Given the anticancer effects of these peptides, which also have potential applications in tumor diagnosis and targeted drug delivery, further preclinical studies to determine their optimal dosage and toxicity would be beneficial.

Ethical Considerations

Compliance with ethical guidelines
This study was approved by the Ethics Committee of Yazd University, Yazd, Iran (Code: IR.YAZD.REC.1402.058).

Funding
This article was extracted from master's thesis of Fatemeh Zamani and Niloofar Nematzadeh, approved by Department of Biology, Yazd University, Yazd, Iran. 

Authors contributions
Conceptualization, study design and study supervision: Reyhane Chamani and Mohammad Mehdi Heidari; Data collection, data analysis and Statistical analysis: Fatemeh Zamani and Niloofar Nematzadeh Soteh; Drafting of the manuscript: Reyhane Chamani and Mehri Khatami; Critical revision of the manuscript for important intellectual content: Mohammad Mehdi Heidari and Ali Moradi; Administrative, technical, or material support: Ali Moradi.

Conflicts of interest
The authors declared no conflicts of interest.

Acknowledgements
The authors are gratefully acknowledge the support of the Research and Technology Deputy of Yazd University for their assistance in conducting this study.



 

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