Research on dihydroartemisinin regulates the proliferation, migration, apoptosis and immune-related molecules of colorectal cancer cells through JAK2/STAT3 signaling pathway

doi:10.3877/cma.j.issn.2095-3224.2025.04.004

Abstract

Abstract:

Objective

This study investigates the effects of Dihydroartemisinin (DHA) on the proliferation, migration, apoptosis, and immune-related molecules of colorectal cancer cells, along with the underlying mechanisms involved.

Methods

Human colorectal cancer cells HCT116 and RKO were treated with 6 umol·L^-1 and 12 umol·L^-1of DHA, respectively. Cell viability was detected using the CCK-8 assay; colony formation assay was used to detect cell cloning; wound healing assay and Transwell assay were used to evaluate cell migration; the Calcein-AM/PI double staining was used to assess changes in the number of dead cells; Western blot analysis was used to detect changes in apoptosis-related proteins Bcl-2 and Cleaved-caspase3, signal pathway proteins JAK2/p-JAK2 and STAT3/p-STAT3, and immune factors PD-L1 and CD47. Additionally, quantitative Realtime PCR(qRT-PCR) was performed to measure the changes of immune factors PD-L1 and CD47 at the mRNA level.

Results

Compared with the control group, treatment of HCT116 and RKO cells with DHA at concentrations of 6.25、12.5、25、50、100 umol·L^-1 for 48 h results in a significant decrease in cell viability with increasing drug concentration and prolonged treatment duration. Cell proliferation was inhibited by DHA treatment (HCT116: 3.021±0.014 vs. 2.449±0.008, t=8.302, P<0.001; RKO: 2.666±0.006 vs. 2.122±0.025, t=11.26, P<0.001). The cell clone formation count was significantly decreased (HCT116: 252±6.11 vs. 161.7±3.253, t=13.02, P<0.001; RKO: 329.4±9.368 vs. 204±9.818, t=9.241, P<0.001), cell migration ability decreased (HCT116: 26.29%±0.947% vs. 15.2%±1.409%, t=6.533, P<0.01; RKO: 30.59%±1.441% vs. 14.77%±0.39%, t=10.6, P<0.001) and cell death (HCT116: 37.54%±2.128% vs. 58.74%±1.498%, t=8.145, P<0.01; RKO: 23.97%±1.203% vs. 34.02%±2.225%, t=3.973, P<0.05) increased. Western blot results demonstrate that DHA treatment leads to a decrease in Bcl-2 protein levels (HCT116: 1.001±0.002 vs. 0.551±0.07, t=6.419, P<0.01; RKO: 1.001±0.002 vs. 0.827±0.013, t=12.98, P<0.001), an increase in Cleaved-caspase3 protein levels (HCT116: 1.001±0.002 vs. 1.344±0.119, t=2.904, P<0.05; RKO: 1.001±0.002 vs. 1.515±0.086, t=5.995, P<0.01), and reductions in PD-L1 and CD47 protein levels (HCT116: PD-L1: 0.999±0.001 vs. 0.829±0.029, t=5.517, P<0.01, CD47: 1.001±0.002 vs. 0.763±0.083, t=4.883, P<0.01; RKO: PD-L1: 0.995±0.007 vs. 0.885±0.021, t=2.867, P<0.05, CD47: 0.991±0.011 vs. 0.562±0.093, t=4.577, P<0.05), along with a reduction in the levels of p-JAK2 and p-STAT3 (HCT116: p-JAK2: 1.018±0.019 vs. 0.678±0.066, t=4.901, P<0.01, p-STAT3: 0.999±0.002 vs. 0.691±0.092, t=3.351, P<0.05; RKO: p-JAK2: 1.018±0.019 vs. 0.475±0.064, t=7.092, P<0.001, p-STAT3: 0.999±0.002 vs. 0.488±0.091, t=6.926, P<0.001). qRT-PCR analysis further shows that DHA treatment results in decreased mRNA expression of PD-L1 and CD47 (HCT116: PD-L1: 1.002±0.042 vs. 0.888±0.019, t=4.052, P<0.05, CD47: 1±0.003 vs. 0.868±0.014, t=9.098, P<0.001; RKO: PD-L1: 1±0.002 vs. 0.671±0.024, t=13.64, P<0.001, CD47: 1.011±0.02 vs. 0.727±0.02, t=10.07, P<0.001). The addition of the JAK2 agonist Coumermycin A1 counteracts the inhibitory effects of DHA on colorectal cancer cells.

Conclusion

DHA can inhibit the proliferation and migration of colorectal cancer cells, promote apoptosis, and regulate immune-related molecules by inhibiting JAK2/STAT3 signaling pathway.

Key words: Colorectal cancer, Dihydroartemisinin, Apoptosis, Immunity, JAK2/STAT3

Ju Huang, Meng Wang, Dong Han. Research on dihydroartemisinin regulates the proliferation, migration, apoptosis and immune-related molecules of colorectal cancer cells through JAK2/STAT3 signaling pathway[J]. Chinese Journal of Colorectal Diseases(Electronic Edition), 2025, 14(04): 319-332.

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References 41

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组别	JAK2	p-JAK2	STAT3	p-STAT3	PD-L1	CD47	Bcl-2
对照组	1.000±0.001	0.999±0.001	1.000±0.002	1.003±0.004	0.999±0.014	0.999±0.001	1.000±0.005
DHA组	0.725±0.031	0.414±0.024	0.706±0.107	0.576±0.0341	0.592±0.145	0.459±0.031	0.515±0.042
DHA+Coumermycin A1组	0.598±0.028	0.903±0.051	0.864±0.104	0.971±0.167	1.032±0.095	1.092±0.133	0.848±0.0299
t值	1.780	6.817	2.200	5.510	6.149	8.837	4.654
P值	ns	<0.001	ns	<0.001	<0.001	<0.001	<0.01

组别	JAK2	p-JAK2	STAT3	p-STAT3	PD-L1	CD47	Bcl-2
对照组	1.000±0.001	0.999±0.001	1.000±0.002	1.003±0.004	0.999±0.014	0.999±0.001	1.000±0.005
DHA组	0.725±0.031	0.414±0.024	0.706±0.107	0.576±0.0341	0.592±0.145	0.459±0.031	0.515±0.042
DHA+Coumermycin A1组	0.598±0.028	0.903±0.051	0.864±0.104	0.971±0.167	1.032±0.095	1.092±0.133	0.848±0.0299
t值	1.780	6.817	2.200	5.510	6.149	8.837	4.654
P值	ns	<0.001	ns	<0.001	<0.001	<0.001	<0.01

组别	JAK2	p-JAK2	STAT3	p-STAT3	PD-L1	CD47	Bcl-2
对照组	1.000±0.001	0.999±0.001	1.000±0.002	1.003±0.004	0.999±0.014	0.999±0.001	1.000±0.005
DHA组	0.674±0.065	0.382±0.050	0.793±0.103	0.354±0.024	0.455±0.0431	0.332±0.019	0.369±0.035
DHA+Coumermycin A1组	0.636±0.115	0.959±0.063	0.950±0.021	1.040±0.015	1.042±0.095	0.905±0.074	0.822±0.060
t值	0.726	11.04	3.002	13.13	11.23	10.96	8.660
P值	ns	<0.001	ns	<0.001	<0.001	<0.001	<0.001

组别	JAK2	p-JAK2	STAT3	p-STAT3	PD-L1	CD47	Bcl-2
对照组	1.000±0.001	0.999±0.001	1.000±0.002	1.003±0.004	0.999±0.014	0.999±0.001	1.000±0.005
DHA组	0.674±0.065	0.382±0.050	0.793±0.103	0.354±0.024	0.455±0.0431	0.332±0.019	0.369±0.035
DHA+Coumermycin A1组	0.636±0.115	0.959±0.063	0.950±0.021	1.040±0.015	1.042±0.095	0.905±0.074	0.822±0.060
t值	0.726	11.04	3.002	13.13	11.23	10.96	8.660
P值	ns	<0.001	ns	<0.001	<0.001	<0.001	<0.001

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