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Effects of the exercise-inducible myokine irisin on proliferation and malignant properties of ovarian cancer cells through the HIF-1 α signaling pathway

Background Exercise has been shown to be associated with reduced risk and improving outcomes of several types of cancers. Irisin −a novel exercise-related myokine- has been proposed to exert beneficial effects in metabolic disorders including cancer. No previous studies have investigated whether irisin may regulate malignant characteristics of ovarian cell lines. Methods In the present study, we aimed to explore the effect of irisin on viability and proliferation of ovarian cancer cells which was examined by MTT assay. Then, we evaluated migratory and invasive ability of the cells via transwell assays. Moreover, the percentage of apoptosis induction was determined by flowcytometery. Furthermore, the mRNA expression level of genes related to the aerobic respiration (HIF-1α, c-Myc, LDHA, PDK1 and VEGF) were detected by real-time PCR. Results Our data revealed that irisin treatment significantly attenuated the proliferation, migration and invasion of ovarian cancer cells. Besides, irisin induced apoptosis in ovarian cancer cells. We also observed that irisin regulated the expression of genes involved in aerobic respiration of ovarian cancer cells. Conclusion Our results indicate that irisin may play a crucial role in inhibition of cell growth and malignant characteristics of ovarian cancer. This findings may open up avenues for future studies to identify the further therapeutic use of irisin in ovarian cancer management.


Introduction
Ovarian cancer (OC), as the most common and lethal gynecologic malignancies, 46 is the fifth cause of cancer-related death in women globally [1,2]. Due to the fact 47 that OC is usually asymptomatic at early stages, most of the patients will 48 ultimately progress to advance stages, resulting in a high rate of mortality [3,4]. 49 Despite recent advances, the lack of effective screening methods and therapeutic 50 modalities are the major hurdles to improve prognoses for management of the 51 disease and the main cause of 5-year survival rate of less than 40% [5]. Thus, 52 exploring novel avenues to improve the status quo is urgently needed. 53 Obesity has been widely reported to be associated with the metabolic diseases 54 including cancer [6][7][8]. Notably, obese people are usually at increased risk of 55 developing cancers due to the higher levels of insulin and insulin-like growth 56 factor-1, which may be responsible for certain tumors promotion [9]. Exercise is 57 one of the several modifiable factors known to reduce the risk of developing 58 human malignancies [10][11][12][13]. It has been also well documented that exercise has 59 many benefits after diagnosis. There is increasingly mounting evidence that 60 exercise relieve many of the common side effects contributed to the conventional 61 cancer therapy among patients, resulting in a better overall quality of life [14][15][16][17]. 62 Besides, a number of observational cohort studies support the view that cancer 63 survivors who had a regular exercise suffered from a lower risk of relapse or 64 cancer-related death after starting treatment regimens [18][19][20].
Recently, skeletal muscle is gaining special interest in terms of releasing various 66 myokines which exert beneficial effects in metabolic disorders. Of these, irisin, a 67 novel promising identified myokine releasing from skeletal muscle following 68 exercise, is believed as a potential therapeutic in a variety of diseases [21,22]. 69 Previous studies have documented the positive association between irisin and 70 body energy expenditure as well as insulin sensitivity [23]. Additionally, irisin 71 has been verified to have a pivotal role in smooth muscle cell phenotype 72 modulation by regulating endothelial cell proliferation, apoptosis and migration 73 [24]. Due to the strong metabolic effects of irisin on several tissue types, it is 74 questionable whether irisin has the ability to modulate malignant features of cells 75 and tissues similar to other myokines [25]. In the past decade, the relationship 76 between irisin and different cancers have been the focus of many studies.

77
Represented data from a recent study found a lower level of serum irisin in 78 patients with breast cancer, while other studies indicated that irisin was 79 significantly increased in gastrointestinal cancer tissues [26][27][28]. Moreover, a 80 number of studies have detected increased irisin immunoreactivity in ovarian, 81 cervix and breast cancer tissues as well as endometrial hyperplasias [29].

82
In the context of cancer metabolism, reprogramming of glucose metabolism is a 83 pervasive microenvironmental event in tumorigenesis. To this end, cancer cells 84 preferentially switch from oxidative phosphorylation (OXPHOS) to glycolysis, 85 even under normal oxygen concentration. This particular metabolic profile -86 defined as Warburg effect-is a key hallmark of many solid tumors including OC 87 [30]. 88 Growing evidence demonstrated that many signal molecules including tumor 89 suppressor genes and oncogenes play substantial roles in conferring metabolic 90 advantages and adaptation to the tumorigenic microenvironment [30]. Of these 91 documented genes, the hypoxia-inducible factor-1α (HIF-1α) is a transcription 92 factor regulating many pivotal pathways in cancerous cells [31].         Table 1.     210 We applied the transwell system to explore the effect of irisin on the migration    significant effect was detected in other two cell lines (Fig.4).

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Ovarian cancer, as the most frequent gynecologic malignancies, is a type of cancer 265 accompanied with hypoxic microenvironment [34][35][36].Exercise is known to be a prominent 266 component of cancer treatment strategies. However, the effect of exercise on tumor 267 oxygenation, hypoxia and vascularization are not fully understood [37].

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The purpose of the current study was to investigate the effects of irisin, as one of the novel hypoxic conditions [41,42]. It is repeatedly reported that HIF-1α remarkably upregulates in 304 OC or borderline tissues in relative to benign tissues [43,44]  subsequently LDHA expression have been reported to be downregulated during exercise [51].

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One additional striking mechanism is the impact of exercise on growth factors such as (VEGF) 329 as a potent angiogenic stimulator. Disregulated of VEGF has been long known linked with the 330 development of almost all tumors leading to distant metastasis, which is one of the main cause 331 responsible for patient mortality [52,53].

332
Considering the aforementioned points, exercise is a master regulator of many genes 333 contributed to cancer development. Our encouraging results in line with previous studies 334 suggest that irisin, as an exercise-derived hormone, may be a potential anticancer agent, 335 warranting the need for more detailed investigations for having better insights about irisin's 336 effects as well as underlying mechanisms in cancer therapy.