br Cinnamaldehyde and oxaliplatin synergistically reversed hypoxia induced
3.4. Cinnamaldehyde and oxaliplatin synergistically reversed hypoxia-induced EMT and stemness of CRC cells
As the result of RT-PCR assay showed, both CA and oxaliplatin had no evident influence on the expression of EMT and stemness markers in normoxia (Fig. 3B and C). When it is under hypoxia, E-cadherin was upregulated by CA and oxaliplatin respectively and more strengthened in combined group. Inversely, mRNA expression of mesenchymal mo-lecules and transcriptional factors exhibited opposite trend (Fig. 5A). Protein levels were accordant as increased E-cadherin and decreased N-cadherin, Vimentin, Snail and Twist expression was evidenced by Western Blot (Fig. 5B). Further spheroid formation study proved that combination of CA and oxaliplatin possesses stronger inhibition on colony forming ability than individual group (Fig. 5C). At mRNA and protein levels, cell stemness markers including CD133, CD44 and Oct4 were gradually reduced in response to CA and oxaliplatin either re-spectively or jointly (Fig. 5D and E). These findings manifested that CA and oxaliplatin can prohibit hypoxia-induced EMT and stemness of CRC Minocycline synergistically.
3.5. Cinnamaldehyde combined with oxaliplatin blocked hypoxia-activated Wnt/β-catenin pathway
We discovered that CA and oxaliplatin were able to prohibit the activation of Wnt/β-catenin signaling by down-regulating β-catenin and Cyclin-D1 under normoxia condition (Fig. 3B and C). Moreover, during the process of hypoxia induction, we found that β-catenin as well as its downstream molecules c-Myc and Cyclin-D1 were upregu-lated at both mRNA and protein levels. In addition, the multifunctional kinase p-GSK3β got increased, contributing to strengthened expression of nuclear β-catenin. However, expression of β-catenin, c-Myc, Cyclin-D1, p-GSK3β and nuclear β-catenin were all downregulated when hy-poxic CRC cells were incubated with CA or oxaliplatin respectively. The inhibitive eﬀect on β-catenin and downstream genes got more sig-nificant in CA + oxaliplatin group (Fig. 6A and C). Besides, immuno-fluorescence assay probed cytoplasmic accumulation and nuclear lo-calization of β-catenin under hypoxia, which was obviously reversed by CA and oxaliplatin (Fig. 6B). We inferred that Wnt/β-catenin signaling is actived under hypoxia condition while CA and oxaliplatin can inhibit the activation synergistically.
3.6. Cinnamaldehyde sensitized colorectal cancer cells to oxaliplatin in vivo
As is shown, CA or oxaliplatin reduced tumor growth campared to control group. Moreover, combination of CA and oxaliplatin repressed the volume and weight of tumors more eﬀectively (Fig. 7A–C). Fur-therly, we verified that the anti-apoptotic marker Bcl-2 was down-regulated while the pro-apoptotic marker Bax was upregulated in CA and oxaliplatin group. The pro-apoptotic eﬀect was more evident in the combined group at both mRNA and protein levels (Fig. 7D and E). In addition, pro-apoptotic proteins containing cleaved Caspase-3 and cleaved PARP were gradually reduced by CA and oxaliplatin alone or jointly (Fig. 7E). Immunohistochemistry study of tumor tissue pre-sented incremental Bax and diminishing β-catenin (Fig. 7F). In brief, we identified that CA sensitized CRC cells to oxaliplatin in xenograft mice model consistent with the in vitro consequences.
Comprehensive treatment based on chemotherapy has become the main strategy for patients with advanced and recurrent colorectal cancer. Oxaliplatin-based chemotherapy is currently the first-line scheme in clinic, which has an obvious superiority in terms of antic-ancer activity. Oxaliplatin forms intra-chain and inter-chain cross-links with DNA through alkylation, thereby inhibiting DNA synthesis and replication to promote apoptosis of tumor cells . However, part of patients develop resistance to oxaliplatin, which seriously restricts the eﬃcacy in the course of treatment. Cinnamaldehyde is one of the active compounds from Cinnamon extracts, which is well established to have anticancer property by producing oxidative stress, inducing apoptosis, regulating cell cycle progression and inhibiting invasion . In ad-dition, researchers have verified that cinnamaldehyde is able to sensi-tize colorectal cancer cells to doxorubicin and 5-Fluorouracil [18,19]. Apoptosis is an active death involving a series of molecular activation and expression in order for cellular homeostasis. Resistance of apoptosis is one of the main sources of hyposensitivity to chemotherapeutics in clinic. In our experiments, we discovered that cinnamaldehyde not only inhibits proliferation, but also enhances the sensitivity to oxaliplatin of CRC cells. We found that cinnamaldehyde increases nuclear chromatin condensation and apoptotic rates induced by oxaliplatin. Additionally, the synergistic eﬀect was also confirmed in CRC tumor xenograft assay, as the volume and weight of tumors were decreased by cinnamaldehyde and oxaliplatin respectively but more eﬀectively in the combined group. Mechanismly, cinnamaldehyde and oxaliplatin promote the ex-pression of pro-apoptosis molecules of CRC cells synergistically both in vitro and in vivo. Additionally, our previous research discovered that