Apigenin Increases Natural Killer Cytotoxicity to Human Hepatocellular Carcinoma Expressing HIF-1α through High Interaction of CD95/CD95L

Natural killer (NK) cell activity is more attenuated in hepatocellular carcinoma (HCC) patients than normal. Hypoxic-inducible factor (HIF)-1α is highly expressed in tumors to maintain their metabolism in a hypoxic environment. The expression of HIF-1α in cancers can lead to cell growth, proliferation, invasion/metastasis and immune escape. Although apigenin, a flavonoid, is known to have various biological activities, it has not been demonstrated in NK cell immune activity in HCC cells. In this study, NK-92 cells were directly cocultured with HCC SK-Hep1 cells for 24 h to evaluate NK cell activity in HCC cells or HCC cells expressing HIF-1α by apigenin. NK cell cytotoxicity to HCC cells expressing HIF-1α was significantly increased, and NK cell-activating receptors, NKG2D, NKp30 and NKp44 were highly expressed. The activating effect of apigenin on NK cells substantially induced apoptosis in HCC cells expressing HIF-1α through high expression of CD95L on the surface of NK-92 cells. Moreover, apigenin excellently inhibited the level of TGF-β1 in a coculture of NK cells and HCC cells. In conclusion, apigenin seems to be a good compound that increases NK cell cytotoxicity to HCC cells by controlling HIF-1α expression.


Statistical Analyses
All data were analyzed using Microsoft Excel. The results were presented as means ± SD, and the comparison of several means was performed by one-way or two-way analysis of variance followed by Fisher's exact test. Differences between groups at a p-value of less than 0.05 were considered significant.

Increase in NK Cytotoxicity through High Secretion of Granzyme B from NK Cells to HIF-1α-Expressing HCC Cells by Apigenin
To identify the increase in NK cytotoxicity to HCC cells by apigenin by LDH-release assay, a secretion of perforin and granzyme from NK cells was examined in a coculture of HCC cells with or without apigenin treatment (50 μM). The concentration of apigenin used in NK cytotoxicity to HCC cells was examined by CCK-8 assay. As shown in Fig. 1A, apigenin did not have a negative effect on NK cell viability while seeming to increase cell proliferation at over 50 μM (*p < 0.05). NK cytotoxicity to target cells showed no change with apigenin treatment ( Fig. 1B; control; 30.96%, apigenin; 28.87%, ns). The secretion of GrzB from NK cells to target cells also showed no significant difference between control and apigenin treatment (Fig. 1C, ns). However, apigenin interestingly increased NK cytotoxicity and the production of GrzB in a coculture of NK and HCC cells when HIF-1α was expressed (NK cytotoxicity; Fig. 1B; CoCl 2 4.68%, CoCl 2 +apigenin 9.99%; *,**p < 0.05)

Decreased Expression of HIF-1α in HCC Cells Cocultured with NK Cells by Apigenin
To recognize whether apigenin inhibits HIF-1α expression, we investigated the expression of HIF-1α in HCC cells cocultured with NK cells using Western blot analysis. As shown in Fig. 3A, the expression of HIF-1α in HCC cells cocultured with NK cells was significantly lowered when treated with apigenin (*,**p < 0.05).

Higher Interaction of Fas (CD95)/FasL (CD95L; CD178) between NK Cells and HCC Cells by Apigenin
The secretion of GrzB from NK cells to HCC cells was significantly increased by apigenin (Fig. 1C). According to recent reports, GrzB stimulates a death receptor on the surface of target cells and subsequently induces the apoptosis of target cells [12]. Thus, we tried to examine a death receptor associated with GrzB. In Figs. 3B and 3C, the expression of CD95 (Fas) significantly increased after treatment with apigenin, and FADD, which is an adaptor protein of the bridge Fas receptor was also highly increased with apigenin treatment. Moreover, CD178 (CD95L) on the surface of NK-92 cells was significantly upregulated by apigenin regardless of HIF-1α expression (Fig. 3C, CD178 + ; CoCl 2 8.1%, apigenin 11.72%, CoCl 2 +apigenin 9.69%, *,**p < 0.05).

Fig. 1. Increase in NK cytotoxicity through high secretion of granzyme (Grz)-B from NK cell to target cell.
(A) NK cell viability on various concentration of apigenin using CCK-8 assay. (B) cytotoxicity and (C) granzyme B secreted from NK cells cocultured with HCC cells with or without treatment with CoCl 2 (250 μM) and apigenin (50 μM). All data were presented as means ± SD from three independent experiments. *p < 0.05 vs non-treated; **p< 0.05 vs CoCl 2 .

Increased CD95-Mediated Apoptosis in HCC Expressing HIF-1α by Expressing High CD95L (CD178) on the Surface of NK Cells by Apigenin
The receptor CD95 binds to CD95L which is implicated in immune homeostasis and immune surveillance and is expressed on the surface of activated T lymphocytes and NK cells [21,22]. Binding CD95L to CD95 stimulates  the activation of FADD, and that triggers apoptosis by the activation of caspase-8. CD95L (CD178) on the surface of NK-92 cells and CD95 (Fas) and FADD in HCC cells increased with apigenin treatment (Fig. 3C). Thus, to verify whether apigenin actually activates the caspases in HCC cells cocultured with NK-92 cells, the expression of caspases (-3,-7,-8 and -9) was investigated by Western blot analysis. The expression of cleaved caspase-8 showed no difference in HCC cells cocultured with NK-92 cells by apigenin but was significant when HIF-1α was expressed (Fig. 4C). Moreover, as shown in Figs. 4A and 4B, the effectors caspase-3 and caspase-7 were significantly activated in HCC expressing HIF-1α cocultured with NK-92 cells by apigenin. The expression of cleaved caspase-9, however, showed no difference between HCC cells in the presence or absence of apigenin (Fig. 4D).

Decrease in TGF-β1 Released from NK-92 Cells Cocultured with HCC Cells Regardless of HIF-1α Expression by Apigenin
Various studies have shown that cytokines in a tumor microenvironment could decide the function of immune cells against cancer cells [23]. Therefore, we examined cytokines secreted from a coculture of NK cells and HCC cells by apigenin treatment. Initially, we identified the level of IFN-γ in the supernatant of a coculture of NK-92 and HCC cells in the presence or absence of apigenin. Unexpectedly, the quantity of IFN-γ in the supernatant of a coculture of NK-92 cells and HCC cells significantly increased when HIF-1α was expressed by treatment with CoCl 2 (250 μM) as compared with no expression of HIF-1α, and apigenin conversely rather decreased the level of IFN-γ ( Fig. 5A; *, **p < 0.05). Furthermore, the expression of HIF-1α in HCC cells cocultured with NK-92 cells significantly increased the level of TGF-β; however, that was remarkably decreased by apigenin ( Fig. 5B; *, **p < 0.05). The cytokines IL-18, IL-21 and IL-10, which can be correlated with NK cell activity, were not greatly affected by apigenin, albeit IL-18 decreased (Figs. 5C-5E; *p < 0.05).

Discussion
In this study, apigenin showed an increase in NK cell cytotoxic effect to HCC cells or HCC cells expressing HIF-1α in vitro. Various cancer types have shown a low frequency and dysfunction of NK cells, and that might promote the metastasis of cancer cells [24]. Moreover, NK cells in patients with HCC commonly present low cytotoxicity and decreased production of interferon (IFN)-γ [11].
Hypoxia is a low-oxygen (O 2 ≤ 1%) condition generated by cancer metabolism and it induces DNA damage. Hypoxia is known to cause genomic changes that tolerate poor nutrition and a hostile microenvironment in tumor cells, and thus tumors are able to survive [25]. HIF-1α is commonly expressed in many tumors, and this inhibits the expression of Bid and pro-apoptotic Bcl-2-family protein [26] and stimulates the expression of survivin, an apoptosis inhibitor [27]. Moreover, the invasion and metastasis of cancer cells is promoted by hypoxia because it can induce the expression of interleukin (IL)-6, platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β [28]. In this study, we first tried to examine the NK cell function in HCC cells when HIF-1α was expressed. HIF-1α expression between NK and HCC cells attenuated the NK cytotoxic effect to HCC cells (Fig. 1B), and also reduced the secretion of granzyme B (GrzB) from NK cells into target cells (Fig. 1C). GrzB is a unique serine protease found in the lytic granules of NK cells and T lymphocytes (CTL). NK cells recognize target cells and then secrete GrzB and perforin into the interspace between them. GrzB in the cytoplasm of target cells leads to the cleavage of caspases, which induces their apoptosis [29]. The results indicated that the expression of HIF-1α in HCC cells decreased NK cytotoxicity. In addition, the expression of NK-activating receptors NKG2D, NKp30 and NKp44 on the surface of NK cells significantly decreased with HIF-1α expression (Fig. 2), which was implicated in the decline of an apoptotic effect of NK cells on HCC cells through a decrease in the cleavage of caspase-3, -7, -8, and -9 (Fig. 4). Recently, many studies have focused on the composition of chemokines or cytokines within the tumor microenvironment. The pro-inflammatory cytokines IL-1β, TNF, and IL-6 can induce the transition of EMT in head and neck cancers [30] and anti-inflammatory cytokine IL-10 can lead to tumor cell proliferation through an induction of STAT3 activation in gastric cancer cells [31]. Also, the presence of IL-10, TGF-β, and prostaglandins in the tumor microenvironment play the role of immunosuppressors that inhibit the anti-tumor activities of NK, T and B cells [32][33][34]. Thus, it is necessary to confirm the level of cytokines, especially that concerned with NK activity. IFN-γ is produced by NK cells by stimulating interleukin(IL)-2, and so we examined the level of IFN-γ produced by a coculture of NK cells and HCC cells. In a coculture of NK cells and HCC expressing HIF-α, the level of IFN-γ was significantly increased (Fig. 5A), contrary to our expectations, while the level of TGF-β1 was substantially increased as well (Fig. 5B). Recently, it has been reported that IFN-γ can lead to EMT transition in pancreatic cells [35] or endometrial cancer cells [36] through the stimulation of MUC4 transcription, which is expressed in an aggressive or metastatic tumor phenotype by the activation of STAT1 [37]. Moreover, several studies showed that IFN-γ signaling enhanced the expression of PD-L1 and induced immune suppression [38]. This evidence fits with our result that an increased level of IFN-γ might be not important in NK cytotoxicity to HCC. However, a high level of TGF-β1 seemed to impair NK activity in HCC (Figs. 1-3). In addition, the level of IL-18 significantly decreased (Fig. 5C), and this was considered to not affect the production of IFN-γ from NK cells but had an inhibitory effect on other factors such as the inflammasome in HCC cells. Our previous study showed that the expression of inflammasome (NLRP3) in HCC could attenuate NK cell cytotoxic ability through the low interaction of NKG2D-MICA/B [39]. This result could mean that a coculture of NK cells and HCC cells might change their environment to induce low NK cell activity by expressing HIF-1α.
The anticancer effect of apigenin is shown in various cancer types containing hepatocellular carcinoma SK-Hep1 and BEL-7402 [40]. According to a recent report, apigenin at a concentration of 25 μg/ml increased NK cell proliferation [20], and the NK immunomodulatory effect of apigenin was recently proved through an induction of cytotoxic granule secretion [41]. Thus, we expected that apigenin could enhance NK cell cytotoxicity to HCC cells. Unexpectedly, in a coculture of NK and HCC cells, apigenin treatment did not increase NK cytotoxicity ( Fig. 1) but incurred a cleavage of caspase-3 (Fig. 4), which could be an anticancer effect of apigenin. Several studies have reported that apigenin disrupted tumor angiogenesis through the inhibition of HIF-1 and vascular endothelial growth factor (VEGF) in several human cancers [42]. The expression of HIF-1α in tumor cells under normoxia resists cytotoxic T lymphocytes (CTLs) [43] and induces immune escape of pancreatic cancer cells from NK cells through the shedding of MICA, which interacts with NK-activating receptor (NKG2D) [44]. In our study, apigenin decreased HIF-1α expression in HCC cells (Fig. 4A), and increased NK cytotoxicity and a secretion of GrzB into HCC cells (Figs. 1B and 1C). The inhibitory effect of HIF-1α has been shown in human pancreatic cancer cells S2-013 and CD18 [45], but not in HCC cells yet. This result indicates that apigenin could restore the functions of NK activity through an inhibitory effect of HIF-1α by apigenin. Furthermore, the expression of NKactivating receptors was significantly increased on the surface of NK cells in a coculture of HCC cells expressing HIF-1α (Fig. 2). More interestingly, apigenin significantly increased the expression of CD178 (CD95L) on the surface of NK cells cocultured with HCC cells expressing HIF-1α (Fig. 3C). Several recent studies have shown that induce fast GrzB-mediated cell death in target cells contacting NK cells and subsequently sustain NK cytotoxic effect through death receptor-mediated killing [12]. The CD95L (FasL; CD178) was expressed at the surface of activated T lymphocytes and natural killer cells [21,22]. This ligand binds their receptor CD95 and subsequently recruits FADD, which is an adaptor protein. Recruiting FADD into CD95 triggers caspase-8 and caspase-10 resulting in apoptosis. Our study showed an activation of caspase-8 in HCC cells expressing HIF-1α cocultured with NK cells in the presence of apigenin (Fig. 4C). In addition, apigenin inhibited excellently the level of TGF-β1 in a coculture of NK cells and HCC cells with or without HIF-1α (Fig. 5B). However, NK activity in HCC was significantly different when HIF-1α was expressed.
Taken together, NK cytotoxic effect on HCC cells was dramatically attenuated by expressing HIF-1α. In particular, the expression of HIF-1α between NK and HCC cells significantly decreased secretion of GrzB from NK cells leading to a decreased killing effect of NK cells toward HCC cells. The attenuation of the NK cell cytotoxic effect on HCC cells expressing HIF-1α, however, could be restored by apigenin. Treatment with apigenin in a coculture of NK and HCC under HIF-1α expression increased the NK cytotoxic effect but had no effect when HIF-1α was not expressed. Interestingly, apigenin increased all expressions of CD178 (CD95L;Fas) on the surface of NK cells in a coculture of HCC cells or HCC cells expressing HIF-1α; however, a difference in the increase in the activation of caspases and GrzB was shown when HIF-1α was expressed. In conclusion, apigenin can be a good compound to increase the NK cytotoxic effect on HCC by inducing high expression of CD95L on the surface of NK cells in a hypoxic condition.