Discussion
GBA is a bioactive sesquiterpene coumarin compound isolated fromFerula species (Bedniak, 1962; Iranshahi et al., 2018).Ferula assafoetida and Ferula Szowitsiana , as rich sources
of GBA, are herbaceous plants of the genus Ferula belonging to
the Umbelliferae family and are distributed throughout central Asia,
eastern Iran, and Afghanistan (Abd El-Razek et al., 2001; Iranshahi et
al., 2007; Yaqoob and Nawchoo, 2016). GBA is an herbal compound with
multiple biological activities including anticancer, cancer
chemopreventive, hepatoprotective, and antiviral activities (Bedniak et
al., 1967; Syrov et al., 1990; Iranshahi et al., 2008; Iranshahy and
Iranshahi, 2011; Kim et al., 2011; Kasaian et al., 2014). GBA exerts its
anticancer activity in association with apoptosis induction,
P-glycoprotein (P-gp) inhibition, and anti-proliferative actions
(Hanafi-Bojd et al., 2011; Kim et al., 2011). The anti-tumor activities
of GBA inspired us to investigate the effects of GBA in MT-2 cell line
to find if GBA can improve the therapeutic efficacy of ATO, as a
potential agent in combination with antivirals and IFN-α in the
treatment of ATL (Kchour et al., 2009), and determine whether the
GBA+ATO combination could be an effective treatment for patients with
ATL.
Results of the present study indicate that GBA is cytotoxic to MT-2
cells in a dose-dependent manner with an IC50 value of 80 µM at 48 h.
The growth inhibitory effects of GBA have been reported in several
studies. Eskandani et al. reported that GBA increases the percentage of
early/late apoptotic cells and attenuates the growth rates of OVCAR-3
human ovarian carcinoma cells in a dose- and time-dependent manner with
an IC50 of 37, 12.1, and 10 µM at 24, 48, and 72 h, respectively
(Eskandani et al., 2015a). In another study, Eskandani et al. showed
that intact GBA and GBA-loaded solid lipid nanoparticles (GBA-SLNs)
inhibited the growth of A549 human lung epithelial carcinoma cells by
upregulation of proapoptotic protein caspase 9 and downregulation of
antiapoptotic protein Bcl-xL (Eskandani et al., 2015b). Kim et al. in
2011 reported that GBA plays its anticancer effects against human
umbilical vein epithelial cells (HUVECs) and Lewig lung cancer (LLC)
cells through anti-angiogenic and anti-proliferative activities (Kim et
al., 2011). Zhang et al. in 2012 demonstrated that GBA preferentially
inhibits the growth of androgen receptor (AR)+prostate cancer cells (with an IC50 of approximately 80 µM at 72 h)
compared to AR- prostate cancer cells by
downregulating the AR levels and consequent inhibition of AR signaling
pathway which is a critical regulator of the G1/S
transition and important for development and progression of prostate
cancer. Additionally, combination of GBA and bicalutamide, an AR
antagonist, showed a greater than additive cytotoxicity in LNCaP
prostate cancer cells (Zhang et al., 2012). In the current study, we
showed that the combination of GBA and ATO was also cytotoxic against
MT-2 cells in a dose-dependent manner and induced the most
cytotoxicity on MT-2 cells after 48 hours. Indeed, the
combination of 20 µM GBA and 4 µM ATO at 48h significantly decreased the
proliferative activity of MT-2 cells compared to each agent alone. In
accordance with our findings, Kim et al. in 2019 reported a significant
dose-dependent cytotoxicity for the combination of GBA and TNF related
apoptosis inducing ligand (TRAIL) in resistant H460/R non-small cell
lung cancer cells (NSCLCs) (Kim et al., 2019). It was also shown that
GBA in combination with nanomicellar curcumin significantly inhibits the
growth of murine C26 and human Caco-2 colon carcinoma cells in a
dose-dependent manner (Jafari et al., 2019). Results obtained in our
study indicate that GBA is able to inhibit MT-2 cells growth
specifically when combined with ATO. Cell cycle analysis also showed
that GBA or ATO alone induced apoptotic cell death and
sub-G1 phase arrest in a low percentage of treated cells
(2.30% and 4.30%, respectively), whereas the combination of GBA and
ATO at the equal concentrations significantly increased the
sub-G1 apoptotic population (55.56%). In line with the
findings of our study, several reports have indicated that GBA increases
sub-G1 apoptotic population. Kim et al. demonstrated
that the combination of GBA (25 or 50 μM) and TRAIL (25 ng/ml) in
resistant H460/R NSCLCs increased distribution of apoptotic cells in
sub-G1 phase to 24.75% and 31.09%, respectively,
compared with 25 μM GBA (6.50%), 50 μM GBA (12.26%), and 25 ng/ml
TRAIL (11.51%) (Kim et al., 2019). Oh et al. have also shown that GBA
at the concentrations of 25 and 50 µM significantly increases
sub-G1 phase cells population in H460 NSCLCs to 7.71%
and 7.92%, respectively, compared with the untreated control (0.17%)
(Oh et al., 2015). By contrast, Kim et al. have detected no apparent
changes in the frequency of GBA-treated LLC cells in the
sub-G1 phase, indicating that GBA could not induce
apoptotic cell death in LLC cells. Nevertheless, cell cycle analysis
showed that GBA inhibited the LLC cells growth by causing an evident
G2/M phase arrest (Kim et al., 2011). Contrary to the
findings of Kim et al. study, it is clear that the GBA+ATO combination
treatment synergistically induced sub-G1 apoptotic
population in MT-2 cells.
Pumping therapeutic agents out of cell, mediated by multiple drug efflux
transporters of the ATP-binding cassette (ABC) family, is a serious
impediment to the effective chemotherapy. Overexpression and enhanced
efflux activity of MDR1/P-gp or ABC sub-family B member 1 (ABCB1) have
been reported in patients with ATL. It has been also reported that
HTLV-1 Tax protein is a potent transcriptional activator of MDR1/P-gp
gene promoter. MDR1/P-gp overexpression endows ATL cells with a drug
resistance phenotype (Lau et al., 1998). Therapeutic agents with the
ability to inhibit the P-gp mediated efflux activity could be used in
combination with existing medicines to increase their potency against
ATL cells. Several studies have investigated the effects of GBA on
MDR-1/P-gp. Studying the effects of GBA on P-gp efflux activity via
rhodamine 123 efflux assay in doxorubicin-resistant MCF7/Adr breast
cancer cells revealed that GBA is a potent P-gp inhibitor that is more
effective in inhibiting the function of ABCB1/P-gp compared to
verapamil, a typical P-gp inhibitor (Hanafi-Bojd et al., 2011). Using
GBA against six Multi-drug resistance clinical isolates ofStaphylococcus aureus showed that GBA has efflux inhibitory
effects on P-gp and its mechanism of action is comparable to verapamil
(Bazzaz et al., 2010). In another study, Kim et al. showed that GBA in
combination with TRAIL inhibits MDR1 efflux activity by repressing the
MDR1 expression in resistant H460/R NSCLCs, thereby leading to enhanced
TRAIL-induced apoptosis induction in NSCLCs (Kim et al., 2019). The
present study confirms previous findings about GBA inhibitory effect on
the functionality of the P-gp efflux pump. Results obtained from efflux
assay showed that the rate of MTX accumulation in MT-2 cells increased
in the presence of GBA. The enhanced accumulation of MTX in GBA-treated
MT-2 cells may be attributed to efflux inhibitory properties of GBA.
Therefore, GBA increased intracellular accumulation of ATO in MT-2 cells
through inhibition of P-gp efflux activity and therefore could overcome
the P-gp mediated Multi-drug resistance and induced apoptotic cell death
in MT-2 cells.
The expression levels of genes involved in cell proliferation and
apoptosis is altered in ATL, resulting in an increase in cell
proliferation and survival and a reduction of apoptosis (Duyao et al.,
1992; Mori et al., 1999; Ariumi et al., 2000; Haller et al., 2000;
Yoshida, 2001; Sun and Yamaoka, 2005; Krueger et al., 2006). In the
present study, we found that the mRNA levels of RelA, CDK4, and c-MYC
significantly decreased after treatment with 20µM GBA. However, when
combined with 4µM ATO, only the expression levels of CDK4 was
downregulated, although, the reduction in CDK4 levels in treatment with
GBA alone was greater than the reduction mediated by the GBA+ATO
combination. Cyclin dependent kinase 4 (CDK4) mediates
G1/S cell cycle progression (Sherr and Roberts, 1995).
In HTLV-1 infected cells, Tax interacts with inhibitors of CDK activity
such as p16INK4A, leading to uncontrolled cell proliferation (Suzuki et
al., 1996; Haller et al., 2000; Yoshida, 2001). GBA-mediated
downregulation of CDK4 might inhibit transition from G1phase of cell cycle to S and therefore induces sub-G1phase cells accumulation. In addition to CDK4, we also observed reduced
expression levels of c-FLIPL and c-FLIPS in cells treated with the
GBA+ATO combination, which might associate with induced apoptotic cell
death in MT-2 cells. Several studies have reported that high expression
levels of c-FLIP supports the cancer cells to elude the
immunosurveillance and promotes the tumor cells growth (Djerbi et al.,
1999; Medema et al., 1999). Tax expressing HTLV-1 infected T cells
express high levels of c-FLIPL and c-FLIPS which contributes to
inhibition of apoptosis in infected cells, blocking the receptor
mediated cell death, evading the host immune response and supports
development of HTLV-1 associated diseases (Krueger et al., 2006; Okamoto
et al., 2006). GBA-induced downregulation of c-FLIPL and c-FLIPS in MT-2
cells treated with the GBA+ATO combination could augment death receptor
mediated apoptosis in MT-2 cells and make them more susceptible to
apoptotic cell death.
The GBA+ATO combination unexpectedly decreased the expression levels of
the tumor suppressor p53 in MT-2 cells. p53 plays a crucial role in
cancer prevention by controlling the cell cycle progression (Ozaki and
Nakagawara, 2011; Xu-Monette et al., 2012). In the current study, a
significant positive correlation was found between the expression of
c-FLIPS and p53. The GBA-mediated downregulation of p53 might be related
with the involvement of p53 in c-FLIPS expression (Bartke et al., 2001).
c-FLIP contributes to apoptosis resistance. Therefore, treatment with
the GBA+ATO combination resulted in downregulation of p53 expression in
MT-2 cells and consequently inhibition of the p53-induced upregulation
of c-FLIPS and therefore suppression of the c-FLIPS apoptosis inhibitory
effects. On this basis, we conclude that apoptosis induction in MT-2
cells seems to be independent of p53 activation. Taken together, GBA+ATO
mediated downregulation of p53 and c-FLIPS might lead to induction of
death receptor-mediated apoptosis in MT-2 cells.