The main finding of our study is that in mice injected with EMT-6 cells, treatment with a combination of GBE and CTX produced better growth inhibition of tumors compared to PBS-treated mice. GBE or CTX alone did not produce a better inhibition on the growth of EMT-6 cells than PBS. Although the results of the survival analysis did not reach statistical significance, a clear upward shift was seen in the survival curves in the GBE + CTX group compared to the PBS controls. Furthermore, the median latency time comparing the GBE + CTX group to the PBS control group with p = 0.06 suggests that statistical significance likely would have been achieved with a larger sample size. Additional assessment of indicators of ROS and apoptotic pathways supported these results. This finding is important because it might offer a treatment regimen for prevention of tumor recurrence.
As expected, MDA levels were higher and SOD activities lower in PBS-treated mice compared to untreated, control mice because cancers cells produce higher levels of ROS [15, 16]. MDA was lower and SOD activity higher in the GBE group compared to the PBS group, demonstrating the antioxidant effects of GBE. CTX showed opposite effects of GBE on MDA and SOD but adding GBE to CTX caused the mice in the combination group to look similar to the GBE group. These comparisons support previous observations that cancer cells and CTX both increase ROS and provides evidence that GBE can reduce ROS with and without CTX present.
Although there is a balance between the benefits and disadvantages of adjuvant antioxidants in chemotherapy, early co-administration of antioxidants with chemotherapeutic agents might be useful in scavenging ROS. The resulting reduction in ROS signaling pathways may, in early stages, suppress tumor growth. Our results showed that GBE + CTX reduced IκB expression and its phosphorylation above that of CTX alone, resulting in decreased activation of the NF-κB signaling pathway and promoting apoptosis. The pro-death response to GBE + CTX was consistent in the increased expression of FADD, Caspase-8, Cyto-C, Bax and Bax/Bcl-2 ratio showing the highest levels in the PBS group, followed by the GBE, CTX and the GBE + CTX groups. A reverse pattern was observed for expression of Bcl-2. These patterns of expression support the hypothesis that GBE acts to promote apoptosis in the early stage of tumor initiation and enhance the effects of CTX.
Downstream of the PI3-K/Akt pathway is p65 where phosphorylation in the IKK-dependent pathway leads to activation of NF-κB, resulting in the promotion of carcinogenesis [8, 17]. Decreased PI3-K/Akt pathway activity promotes autophagy-mediated cell death [18, 19]. Autophagy is initiated in part by genotoxic stress with an increased expression of p53 [20–22], and our results showed an increase in p53 in the GBE + CTX group compared to the PBS group.
In summary, in our study CTX and GBE was administered to the mice after the tumor cells were transplanted into the mice after only 24 hours, which was the initial stage of tumor growth. There is inconsistency regarding whether antioxidation should be used in tumor treatment . Tumor weight was significantly decreased by CTX + GBE. The mechanisms underlying this change are that GBE + CTX significantly inhibited the activation of NF-kB, upregulated the proapoptotic and P53 genes, and downregulated the PI3K/Akt pathway, which are implicated into the expansion of cancerous cells. It has been reported that ROS will promote the growth of tumor cells by triggering one of the cell growth signaling pathways, NF-kB. GBE is able to remove ROS which can activate NF-kB, as shown in our findings. The expression and phosphorylation of NF-kB were both significantly suppressed in the GBE + CTX group. Moreover, ROS is also able to promote the expansion of cancerous cells by modifying the genes related to apoptosis and to upregulate antiapoptotic genes and downregulate proapoptotic proteins via PI3K/AKT and ERK/MEK pathways . GBE + CTX is also able to increase the expression of genes related to apoptosis Bax, FADD, Caspase-8, and Cyto-C. Furthermore, PI3K/AKT was significantly decreased by GBE + CTX.
The strength of the present study is that we conducted an experiment to investigate whether or not, when the tumor cells was already present in the body, at the early stage of tumor development, chemotherapy and antioxidation would produce a better inhibition on the growth of tumor cells. The findings of our study suggest that CTX alone would not inhibit the growth of EMT-6 cells better than PBS, or GBE, indicating that when tumor cells were present, chemotherapy would not be potent enough to kill the tumor cells inside body. After surgery, antioxidation and chemotherapy might be helpful to fight the recurrence or slow the growth of tumor cells, our findings are in accordance with Thomson’s et al’ .
A weakness of the present study is that the tumor cells were injected into the mice; the procedure may not simulate the development of tumor onset in the real situation. All of the mice had developed palpated tumors on or before day 10 in our experiment. Had we continued the trial period, we might have observed a significant difference in tumor weights between CTX and GBE + CTX treated groups.