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consequences of FCM enhibited (Fig. 1D). At the protein levels, ox-aliplatin eﬀectively upregulated Bax, cleaved Caspase-3, cleaved PARP and downregulated Bcl-2, which were evidently reversed while Dynasore were under hypoxic condition. It is noticeable that, the expression of HIF1α was strengthened in hypoxia but presented no significant change in response to oxaliplatin (Fig. 1E). The above results implied that hypoxia is able to impair the pro-apoptosis eﬀect in CRC cells.
3.2. Hypoxia induces EMT and stemness in CRC cells
In the hyoxic environment, we observed decreased epithelial mo-lecule E-cadherin and increased mesenchymal markers as well as transcriptional factors including N-cadherin, Vimentin, Snail and Twist at both mRNA and protein levels (Fig. 2A and B). The spheroid for-mation rate of cells under hypoxia was apparently higher than those in normoxia group (Fig. 2C). Meanwhile, mRNA expression of stemness markers containing CD133, CD44 and Oct4 got upregulated in hypxia (Fig. 2D), while the result of Western Blot assay showed similar ten-dency at protein levels (Fig. 2E). These consequenses proved that CRC cells exprienced EMT and acquired stemness during hypoxia.
3.3. Cinnamaldehyde sensitized colorectal cancer cells to oxaliplatin
CRC cells treated with cinnamaldehyde and oxaliplatin separately or synergistically put up diﬀerent viability under both normoxia and hypoxia. During our past research, 40 μg/ml of CA was confirmed as the concentration according to the IC50 value . At present, we found that CA and oxaliplatin could synergistically inhibited CRC cell pro-liferation under normoxia, by way of inducing expression of apoptosis molecules (Fig. 3). We also discovered that 40 μg/ml of CA inhibited proliferation of hypoxic CRC cells, just as the eﬀect caused by ox-aliplatin individually. However, CA combined with oxaliplatin de-creased cell viability more eﬃciently as the MTT assay showed (Fig. 4B). Hoechst 33258 experiment showed that CA + oxaliplatin group has more distinct phenomenon of nuclear chromatin condensa-tion than single or control groups (Fig. 4C). Apoptotic rate of CRC cells treated with CA + oxaliplatin was higher than separately treated groups (Fig. 4D). Additionally, anti-apoptotic marker Bcl-2 got down-regulated while apoptosis protein Bax, cleaved Caspase-3 and cleaved PARP1 got upregulated in response to CA and oxaliplatin synergistically (Fig. 4E). Besides, both protein and mRNA levels of HIF-1α was not changed by CA or oxaliplatin (Fig. 4E and F). All the results indicated that CA is able to enhance the sensitivity to oxaliplatin of hypoxic CRC cells.
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 cells 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.