The current study provides, to our knowledge, the first mechanistic evidence for the ability of camel milk to significantly inhibit the growth and proliferation of human liver HepG2 and breast MCF7 cancer cells through apoptotic- and oxidative stress-mediated mechanisms. In this study, molecular mechanisms underlying camel milk-induced cell growth inhibition and apoptosis were measured on the following perspectives: (a) the activation of caspase-3 at the mRNA and activity levels, (b) the activation of DR4, (c) the accumulation of intracellular ROS, and (d) the role of MAPK signaling pathways.
Apoptosis is a tightly regulated process under the control of several signaling pathways, such as caspases and mitochondrial pathways [4
]. Particularly, caspase-3 is an important proapoptotic protein within the extrinsic and intrinsic apoptotic pathways [3
]. Activation of caspase-3 plays a central role in the initiation of apoptosis, which requires the activation of initiator caspases, such as caspase-8 or -9, in response to proapoptotic signals [3
]. Induction of apoptosis with generation of ROS by cancer chemoprotective agents, such as doxorubicin [2
], not only induces cancer cell death but also causes DNA damage and genomic instability [18
]. However, most of these cancer chemoprotective therapies are cytotoxic and their use is associated with toxicities. Thus, the development of new chemopreventive agents able to inhibit cell proliferation and induce apoptosis in cancer cells but with less or no side effects is important and anticipated.
Previous study from our laboratory have reported the ability of camel milk to inhibit the expression of CYP1A1
, a cancer-activating gene, and to induce the expression of NQO1
, a chemoprotective gene, in murine hepatoma Hepa 1c1c7 cells [8
]. However, the chemoprotective effect of camel milk against human cancer cells had not been examined before. Therefore, to model the in vivo
situation, human hepatoma HepG2 and breast cancer MCF7 cell lines were used in the current study to predict human responses to camel milk by investigating the capacity of camel milk to inhibit HepG2 and MCF7 cells growth and proliferation and explore the role of apoptosis in camel milk-mediated effect.
Initially, we have assessed the potential effect of camel milk to suppress the growth and proliferation of HepG2 cells using the MTT assay since this assay has been demonstrated to correlate with clinical outcome [19
]. We demonstrated here that camel milk, but not bovine, was able to significantly suppress HepG2 cell proliferation and growth (). Importantly, the camel milk-mediated effect in HepG2 cells is attributed to induction of apoptotic signaling pathway, as evidenced by activation of caspase-3 mRNA and activity levels without changes in the expression of p53 and Bcl2 (), suggesting a p53-independet mechanism. Although p53
gene was the first tumor suppressor gene linked to apoptosis, DNA-induced apoptosis was shown in cell lines lacking p53 expression or mutated p53 [20
]. Furthermore, studies using transgenic and knockout mice have reported the ability of several chemotherapeutic agents, such as curcumin [21
] and doxorubicin [2
], to induce apoptosis through a p53-independent pathway.
Cell surface death receptors have been identified as major pathway in apoptotic induction [22
], which involves the ligation of the TNF/Fas-receptor with its ligand followed by caspases activation [24
]. Therefore, we have tested whether DRs are involved in camel milk-mediated effects by measuring the expression of DR4. In this regard, we have shown here that induction of DR4 mRNA in response to camel milk significantly contributes to the activation of caspase-3. Our results are in agreement with previous observations demonstrated that antitumor agents, such as ET, doxorubicin, Ara-C, 5-FU, and cis-platinum, or irradiation can upregulate the expressions of DR4 and DR5 in acute leukemia, multiple myeloma, and solid tumor cell lines [23
Activation of caspase-3 has also been shown to be mediated through mitochondrial ROS production [26
]. Our data provide evidence that the apoptotic cell death induced by camel milk in HepG2 cells is associated with ROS generation. This is in agreement with previous findings that ROS per se
are potent inducers of apoptosis [27
]. Taken together, it would suggest that induction of ROS in response to camel milk triggers caspase-3 activation which is a direct effect and is not caused by a decrease in the levels of Bcl-2, a protein possesses antioxidant function and blocks ROS production [28
], as no changes in its mRNA expression was observed (). Importantly, similar pattern of caspase-3, DR4, and HO-1 mRNAs induction was observed in human breast cancer MCF7 cells (), suggesting that camel milk-mediated effect is not specific to HepG2 cells. Taken together, these results indicate that HepG2 and MCF7 cells are susceptible to caspase-3-induced cell death, as overexpression of caspase-3 induced direct cytotoxicity to the cells.
Mechanistically, we have shown here that the ability of camel milk to induce caspase-3 gene is a transcriptional mechanism. This was supported by the ability of the transcription inhibitor, Act-D, to significantly block the newly synthesized caspase-3, but not existing, mRNAs (), suggesting a requirement of de novo
RNA synthesis for the induction of caspase-3 mRNA by camel milk [29
]. On the other hand, MAPK cascades play important roles in cell death and survival signaling [30
]. Thus, we have examined and compared their role in casepase-3 mRNA expression by camel milk using specific inhibitors of JNK, ERK, and p38 MAPKs. Inhibition of JNK and p38 MAPK significantly decreased activation of caspase-3 mRNA in HepG2 in response to camel milk, whereas inhibition of ERK signaling pathway enhanced the camel milk-induced activation of caspase-3 mRNA. These finding support previous observations that ERK plays a role in cell survival [32
] and confirm the proapoptotic effect of JNK [4
Although the potential mediators in camel milk involved in apoptosis were not examined in this study, several previous studies have reported that camel milk contains considerably higher amounts of antioxidant vitamins, such as E and C, in comparison to bovine milk [33
], lyzosomes [34
], lactoferrins [34
], and immunoglobulines [35
]. Among these mediators, lactoferrin, an iron-binding glycoprotein, is known to exert in vitro
and in vivo
antitumor activity [36
]. In this context, it has been recently reported that lactoferrin inhibits the development of cancer through inhibiting CYP1A1 activation in 7,12-dimethylbenz[a]anthracene (DMBA-) induced hamster buccal pouch carcinoma model. Taken together, the results obtained from our laboratory and previously published reports, we speculate that lactoferrin could be responsible for camel milk-mediated effect.
In conclusion, to our knowledge, this will be the first study investigating the molecular mechanisms that govern the effect of camel milk on human cancer cells. This study clearly demonstrated that camel milk induces apoptosis in HepG2 and MCF7 cells through apoptotic- and oxidative-stress-mediated mechanisms. These results are of potential clinical significance to humans in that it uncovers the molecular mechanism involved and could explain the anecdotal evidence for the successful use of camel milk in the treatment of various medical conditions.