The co-expression of ghrelin and GHS-R has previously been observed in various neoplasms and related cancer cell lines in humans (for review see:
]). It may suggest their role in neoplastic cell growth and development through autocrine/paracrine mechanism. However, as far as we realize, there are no published reports regarding similar data on tumors in domestic animals. This is the first report of ghrelin and its receptor expression in canine mammary tumor tissues and cell lines isolated from the canine primary carcinomas (CMT-W1 and CMT-W2) and from their lung metastases (CMT-W1M and CMT-W2M).
We have demonstrated the expression of ghrelin in benign and malignant canine mammary tumors (Figures
) and in all of the examined cancer cell lines (Figures
). Interestingly, we observed significantly higher expression of ghrelin in the distant metastases than in the primary carcinomas, which suggest that ghrelin might be involved in mechanism of metastasis (Figure
). A recent study on human colorectal cancers indicated that ghrelin might initiate malignant transformation and might contribute to the cancer spread
]. It was also hypothesized that ghrelin mediates the physiological responses of tissues which surround the tumor, to malignant changes
The presence of GHS-R mRNA has been previously detected in all of the examined canine cancer cell lines
]. Similarly to mRNA expression patterns, GHS-R protein showed significantly higher expression in cell lines isolated from lung metastases of mammary cancer (CMT-W1M and CMT-W2M) what may indicate its role in metastasis (Figures
). Moreover, we have demonstrated that expression of ghrelin receptor varies in canine mammary tumors depending on the histological grade of malignancy (Figure
). The GHS-R expression level was higher in well or moderately differentiated cancers of the 1st
and the 2nd
grade of malignancy compared to poorly-differentiated cancers of the 3rd
grade of malignancy. This intriguing finding overlaps with those reported previously, that expression and functionality of specific binding sites for GHS (growth hormone secreatgogues
) in various types of breast and thyroid carcinomas was maintained in better-differentiated tumors and was decreased in less-differentiated neoplasms
]. Similar expression patterns of GHS-R were observed in human gastric and colorectal cancers
], where the GHS-R expression level was lowest in undifferentiated tumors and correlated inversely with histological grade of malignancy and TNM stage. Moreover, patients with more weight loss showed lower expression of GHS receptor
]. Therefore, decreased ghrelin receptor expression seemed to correlate with poor prognostic factors such as poor differentiation, advanced stage and malnutrition.
The next step of this study was to assess a role of ghrelin in cancer cell biology. Our results revealed that ghrelin stimulates canine mammary cancer cells proliferation and inhibits apoptosis, possibly via an autocine/paracrine mechanism. Although the role of ghrelin in cancer cells was investigated in multiple experiments using human and rat cancer cell lines, the results were equivocal. Some investigators have found ghrelin as antiproliferative factor
], but other have found it a growth stimulator of cancer cells
]. Cassoni and co-workers described an inhibition of cell proliferation in human thyroid, breast and lung cancer by ghrelin
]. Authors found that ghrelin given for 24 hrs at relatively high doses (100 or 1000 nM) decreased cell proliferation. However, after incubation of these cells with ghrelin for 96 hrs, the effect was abolished
]. Volante et al.
] have confirmed antiproliferative effect of ghrelin in thyroid cancer cells showing significant growth reduction after 48 hrs and 96 hrs of incubation with ghrelin at the concentration of 100-1000 nM. Interestingly, Cassoni et al.
] have showed dose-dependent effect of ghrelin treatment in prostate cancer cells. Contrary to the mentioned reports the pro-proliferative effect of ghrelin in several human cancer cell lines has also been well documented. Ghrelin at doses ranging from 1 to 10 nM, which encompasses normal circulating ghrelin levels increased cellular proliferation after 72 h of incubation in breast and prostate cancers
]. Ghrelin treatment significantly stimulated cells growth in the highly metastatic (MDA-MB-435) breast cancer cell line up to 36% above control. The most significant proliferative response to ghrelin treatment occurred at the concentrations of 10 and 100 nM in the MDA-MB-231 cell line, and 1–10 nM in the MDA-MB-435 cell line
]. Thus, these results show that ghrelin given in high doses inhibits cancer cell proliferation, whereas given at low doses increases proliferation. Our results also indicated that ghrelin given at the dose of 1 or 10 nM increased canine mammary cancer cells proliferation in three of four examined cell lines (Figure
), whereas higher doses caused not significant effect also in three of four examined cell lines. In one cell line, ghrelin did not cause any affect.
To clarify the role of ghrelin in cell proliferation the cell cycle analysis was conducted
]. This study revealed that although ghrelin did not change the proportion of cells in the G0/G1, S or G2/M phases of cell cycle, it caused a decrease in the number of apoptotic cells in sub-G0 phase. In fact ghrelin was reported to be the antiapoptotic factor in colonic cancer cells
] as well as in many other types of cells like adipocytes
], osteoblastic cells
], or human endothelial cells
The antiapoptotic activity of ghrelin is also supported by our findings (Figure
). We have showed that ghrelin decreases the number of apoptotic cells in all of the examined canine mammary cancer cell lines (in all of the examined cell lines ghrelin treatment at a dose of 1 nM significantly decreased apoptosis, whereas in two cell lines incubation of cells with the ghrelin concentration of 10 nM also decreased the number of apoptotic cells). Because in CMT-W1M cell line we observed a significant increase in number of apoptotic cells after the treatment with ghrelin inhibitor, our next step was to knock-down the ghsr gene to check if the toxic effect was caused by the inhibitor itself or by blocking the GHS-R. Our results showed that treatment of CMT-W1M cell line with ghsr-specific siRNA also increased the number of apoptotic cells (early apoptotic) (Figure
). An increase in number of apoptotic cells was slight, however significant, which can be explained by efficacy of gene silencing. In our experiment only 85% of cells remained transfected.
Recently, a few studies have focused on the involvement of ghrelin in cancer cells motility and ability to metastasis. Duxbury and co-workers
] investigated the role of ghrelin in metastatic potential of poorly differentiated human pancreatic cancer cells. They found that ghrelin at 10 nM concentration increased not only the proliferation rate of cancer cells but also cellular motility and invasiveness (even up to 60%). Another study showed the promotion of proliferation and invasiveness of human colorectal cancer cells by ghrelin in an autocrine and paracrine manner. This effect was almost completely abolished when the cells were pre-treated with either GHS-R antagonist (D-[Lys3]GHRP6) or a neutralizing ghrelin – specific antibody
]. Furthermore Dixit et al.
] revealed that ghrelin acting via functional GHS-R1a caused increase in intracellular calcium mobilization and led to actin polymerization and membrane ruffling, which resulted in an increase of migration and invasion of carcinoma cells.
Our study also showed a role of ghrelin in cancer cells migration and invasion. We have demonstrated that ghrelin given at the concentration of 100 nM increased the migration ability of canine carcinoma cell lines CMT-W1 and CMT-W1M, evaluated by wound healing test (Figure
). Moreover ghrelin given at the concentration of 1 and/or 10 nM significantly increased migration and invasion of all the examined canine carcinoma cell lines (Figure
The fact that the examined carcinoma cell lines respond in different manner to the various ghrelin doses may be related with differences in endogenous ghrelin production levels. Differences between effective ghrelin doses that stimulate cells proliferation or survival and doses that increase cells migration can be explained by the fact that these different cellular activities are mediated by different cellular pathways. Literature data show that ghrelin increases cellular migration acting on CaMKII, AMPK, and NF-kB pathways
], whereas increases cellular proliferation and survival acting on ERK1/2
]. Thus, probably various ghrelin doses are required to activate various cellular pathways.