Artemisinin inhibits colorectal cancer progression via the FAM83A-AS1/HIF-1α axis

Introduction

Colorectal cancer (CRC) represents a major malignancy that threatens the life and health of individuals in China and imposes a substantial societal burden. Recent data indicate that approximately 387,600 new cases of CRC occurred in China, ranking third among all malignant tumors in incidence (1). The prognosis of CRC is generally poor, as many patients are diagnosed at an advanced clinical stage. Previous studies have reported a 5-year relative survival rate of 90% for stage I disease, whereas the 5-year relative survival rate for stage IV disease with distant metastasis was only 14% (2). Currently, the primary treatment modalities for patients with CRC include surgical resection, radiotherapy, and chemotherapy. Research on targeted therapies has demonstrated favorable therapeutic effects, highlighting the importance of identifying biomarkers that can predict prognosis and influence clinical outcomes in patients with CRC.

Traditional Chinese medicine (TCM) has been used for thousands of years in disease management. Owing to its systemic regulatory effects on the human body and relatively low toxicity, research on its application in various diseases, including CRC, has increased globally (3). Prior studies have demonstrated that TCM inhibits cell proliferation, induces apoptosis, and induces cell cycle arrest (4,5). TCM has been reported to enhance immune function, regulate intestinal flora, and modulate cellular growth (6,7). TCM inhibits the expression of HIF-1α and MMP2, as well as hypoxia-induced reactive oxygen species (ROS) production, migration, and vasculogenic mimicry formation (8). TCM alleviates gastrointestinal adverse reactions associated with anticancer therapies, including diarrhea, nausea, and vomiting, reduces the incidence of myelosuppression and cardiotoxicity, and contributes to the prevention of chemotherapy-induced peripheral neuropathy and radiation-induced pneumonitis (9). It was also indicated that TCM inhibited cell proliferation, induced apoptosis, and induced cell cycle arrest at the G0/G1 phase (4).

Artemisinin is a sesquiterpene lactone compound containing an endoperoxide bridge, extracted from Artemisia annua L. (Compositae), and is associated with relatively low toxicity (10). It has been reported to exert immunosuppressive, anti-schistosomal, antiviral, and anti-inflammatory effects. Numerous studies have demonstrated its potential in antitumor applications (11). Drug repurposing of established agents may substantially reduce the cost associated with drug development. The anticancer mechanisms of artemisinin include inhibition of proliferation through disruption of the cell cycle, induction of DNA damage mediated by ROS, promotion of apoptosis, inhibition of angiogenesis, modulation of immune responses, and enhancement of radiosensitivity (12,13). In studies of CRC, increased heme levels through the use of aminolevulinic acid, a precursor in heme biosynthesis, significantly enhanced the cytotoxic effects of artemisinin on CRC cells (14). Previous studies have indicated that the use of LA and TRFi, as endogenous blood components, may achieve enhanced antitumor activity in patients with cancer following artemisinin administration, without detectable toxicity to normal cells (15). Currently, studies exploring the role of artemisinin in CRC remain limited. Preliminary experimental findings demonstrated that artemisinin inhibited CRC progression through direct regulation of aerobic glycolysis. The aim of this study was to further elucidate the mechanisms by which artemisinin inhibits the development of CRC cells, thereby providing a theoretical basis for its application in the treatment of CRC. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0495/rc).

Methods

Participants

A total of 72 CRC tissue samples, corresponding adjacent non-tumor tissue samples, and serum samples were collected from patients treated at The Fourth Hospital of Hebei Medical University. The mean age of the patients was 57.25 years; 52 (72.2%) were male and 20 (27.8%) were female. Clinical staging indicated that 9 patients were classified as stage I–II, whereas 63 patients were classified as stage III–IV. Lymph node metastasis was not observed in 10 patients, while 62 patients presented with lymph node metastasis. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Medical Ethics Committee of The Fourth Hospital of Hebei Medical University (No. 2022KY112). The informed consent for the use of the patients’ samples (tissue/blood samples) in this study has been obtained.

Cells

The human CRC cell lines HCT-116 and LoVo were obtained from Shanghai Zhongqiao Xinzhou Biotechnology Company and maintained in RPMI-1640 medium (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS; PAN, Adenbach, Germany) at 37 °C in a humidified incubator with 5% CO₂. Cells were seeded into culture plates prior to transfection. Gene knockout lentiviruses purchased from Jikai Gene were used to establish stable cell lines in these human CRC cell lines.

si-FAM83A-AS1: 5'-CCG GCC TTG GGT CTG GAA ACT GTT ACT CGA GTA ACA GTT TCC AGA CCC AAG GTT TTT TG-3'; si-NC:5'-UUC UCC GAA CGU GUC ACG UTT-3'.

Quantitative real-time PCR

Total RNA was extracted from CRC tissue samples, serum, and cell samples using TRIzol. First-strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). Quantitative real-time PCR was performed using the QuantiNova SYBR^® Green PCR Kit (Qiagen, Hilden, Germany). Human GAPDH and U6 snRNA were used as internal controls for FAM83A-AS1, HIF-1A, PKM2, E-cadherin, and N-cadherin, respectively. Gene expression levels were calculated using the 2^−△△CT method. The primer sequences were as follows: FAM83A-AS1: forward primer 5'-GTGTGAGGATGTCCCTGGAT-3'; reverse primer 5'-CCCCAAACAATCATGAGGAG-3'. E-cadherin: forward primer 5'-CGACCCAACCCAAGAATCTA-3'; reverse primer 5'-AATGGCAG GAATTTGCAATC-3'. N-cadherin: forward primer 5'-CCACAGCTCCACCATATGACT-3'; reverse primer 5'-CCCCAGTCGTTCAGGTAATC-3'. U6: forward primer 5'-CTCGCTTCG GCAGCACA-3'; reverse primer 5'-AACGCTTCACGAATTTGCGT-3'. GAPDH: forward primer 5'-AGGTGAAGGTCGGAGTCAACG-3'; reverse primer 5'-AGGGGTCATTGATGGCAACA-3'.

Immunohistochemical analysis

Immunohistochemical staining was conducted to determine the expression of HIF-1α in paraffin-embedded CRC tissue sections and adjacent non-tumor tissues. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 10 minutes. Antigen retrieval was conducted in 10 mM sodium citrate buffer (pH 6.0) at 98 °C for 9 minutes, followed by incubation with 2% normal serum. The slides were sequentially incubated with a primary monoclonal anti- HIF-1α antibody (1:800, 4053S; Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C, followed by incubation with a biotinylated secondary antibody for 60 minutes at 37 °C and ABC reagent for 45 minutes at 37 °C. Visualization of target proteins was achieved using 0.5% 3,3'-diaminobenzidine, and counterstaining was performed with hematoxylin.

Mitochondrial transmembrane potential (MTS) assay

Cell viability was assessed using an MTS assay kit. Treated cells were seeded into 96-well plates at a density of 2×10³ cells per well. Subsequently, 20 µL of MTS reagent (500 µg/mL) was added at 0, 12, and 24 hours following cell attachment. After incubation for 2 hours in a humidified incubator, absorbance was measured at 492 nm using a microplate reader. All experiments were performed in triplicate.

Transwell assays

Cells were treated and seeded into 24-well Transwell chambers (8 µm pore size; Corning, USA) in serum-free medium and incubated for 24 hours. The chambers were pre-coated with Matrigel. Following incubation for 48 hours, the invaded cells were fixed and stained with crystal violet. Images were obtained using an inverted microscope.

Extracellular acidification rate

The extracellular acidification rate was measured using the Seahorse XF Glycolytic Rate Assay Kit (Seahorse Bioscience, Agilent, City, Country, 103344-100), in accordance with the instructions provided by the manufacturer for the Seahorse XFe96 analyzer (Seahorse Bioscience, Agilent).

Western blot

Total protein was extracted using radioimmunoprecipitation assay buffer. Protein concentrations were subsequently determined, after which samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk and then incubated overnight at 4 °C with anti-PKM2, anti-HIF-1α, anti-E-cadherin, and anti-N-cadherin antibodies. GAPDH (Abcam, City, Country) served as the internal control.

Xenograft tumor model

Four-week-old male BALB/c-nu mice (n=10; body weight, 13–14 g) were obtained from Beijing HFK Biosciences Co., Ltd. Following a 7-day acclimatization period, a subcutaneous xenograft tumor model was established by injection of 100 µL of HCT-116 cell suspension (5×10⁶ cells) into the right hind limb. Seven days after cell inoculation, the mice were randomly assigned to two groups: a control group (corn oil) and an artemisinin-treated group (25 mg/kg, administered by gavage and dissolved in corn oil). Treatment was administered daily for 15 days. On the seventh day following the final treatment, the mice were euthanized by cervical dislocation. Tumor tissues were subsequently collected and analyzed using immunohistochemistry (IHC). Experiments were performed under a project license (No. 2022065) granted by the Animal Welfare Ethics Committee of The Fourth Hospital of Hebei Medical University, in compliance with institutional guidelines for the care and use of animals.

Statistical analysis

All statistical analyses were performed using SPSS 22.0 software (SPSS, Inc., USA). Two-sided tests were used to determine statistical significance. Comparisons of rates between HIF-1α-positive and HIF-1α-negative groups across clinical characteristics were conducted using the chi-square test.

Survival rates were assessed using Kaplan-Meier analysis, and comparisons between groups were performed using the log-rank test. A value of P<0.05 was considered statistically significant for all analyses.

Results

Artemisinin inhibited proliferation, migration, cell cycle progression, and glycolysis in CRC cells

Preliminary experimental findings indicated that artemisinin reduced CRC progression through inhibition of glycolysis. To further investigate the effects of artemisinin on CRC cell function, the concentration of artemisinin was initially assessed. A concentration of 20 µM significantly inhibited cell proliferation (Figure 1A). In migration assays, a significant reduction in the migration rate of CRC cells was observed following artemisinin treatment (Figure 1B). Flow cytometric analysis demonstrated that the proportion of cells in the G1 phase of the HCT-116 cell cycle increased from 55% to 61% after treatment with artemisinin (Figure 1C). No significant changes in oxygen consumption were observed following artemisinin treatment in mitochondrial metabolism. In glycolysis assays, lactic acid levels were reduced after artemisinin treatment (Figure 1D).

Figure 1 Artemisinin suppresses cell proliferation, migration, cell cycle, and ECAR in vitro. (A) Cell proliferation was detected by MTS assays after artemisinin treated. (B) Transwell assays were performed after artemisinin treated. (C) The cell cycle of colorectal cancer cells was assessed by flow cytometry. (D) The effect of Artemisinin on mitochondrial metabolism rate and glycolysis were tested by OCR and ECAR in colorectal cancer cells. *, P<0.05. ECAR, extracellular acidification rate; MTS, mitochondrial transmembrane potential; OCR, oxygen consumption rate.

Mechanism of artemisinin regulating CRC

Artemisinin target proteins were obtained from the TCMSP (https://tcmsp-e.com/) and PharmMapper (http://www.lilab-ecust.cn/pharmmapper/submitfile.html) databases, while CRC-related genes were retrieved from the GeneCards and OMIM databases. A total of 96 proteins associated with CRC were identified as potential targets of artemisinin (Figure 2A). These 96 genes were subsequently analyzed using the STRING database and visualized with Cytoscape software. Gene color and size represented the degree of connectivity, with HIF-1α, CCND1, VEGFA, and ESR1 identified as key proteins within the network (Figure 2B). The MCODE plug-in was then applied to construct three hub protein networks. The largest network was primarily composed of cell cycle-related proteins, including HIF-1α, cyclin D1, CDK4, and ZEB1 (Figure 2C). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the target genes indicated that artemisinin was mainly associated with the ubiquitin pathway and DNA binding-related pathways in CRC (Figure S1).

Figure 2 Network pharmacological mechanism of artemisinin regulating colorectal cancer. (A) Venn diagram of the Artemisinin targets. (B) A PPI network of artemisinin targeting proteins. (C) Networks constructed from the PPI network of the target proteins by MCODE. (D) Molecular docking of the artemisinin with the core targets. (E) Expression levels of E-cadherin, N-cadherin, PKM2, HIF-1α, cyclin D1, and CDK4. *, P<0.05. HIF-1α, hypoxia-inducible factor-1α; PPI, protein-protein interaction.

Molecular docking analysis demonstrated that artemisinin formed hydrogen bonds with the 407th THR residue and the 409th ILE residue of the HIF-1α protein, with a binding free energy of −4.4 kcal/mol. Artemisinin formed hydrogen bonds with the 174th and 211th ALA residues and the 181st HIS residue of the Cyclin D1 protein, with a binding free energy of −7.4 kcal/mol. In addition, two hydrogen bonds were formed between artemisinin and the 191st TYR residue of the CDK4 protein, with a binding free energy of −7.7 kcal/mol (Figure 2D). To further validate the mechanism by which artemisinin regulated metastasis and cell cycle progression in CRC cells, the expression of related proteins was assessed. The results indicated that artemisinin reduced the expression levels of cyclin D1 and CDK4 (cell cycle-related proteins), HIF-1α and PKM2 (glycolysis-related proteins), and N-cadherin (metastasis-related protein) (Figure 2E).

Artemisinin promoted the degradation of HIF-1α protein through the proteasome pathway

To determine the mechanism by which artemisinin regulated HIF-1α protein levels, cells were treated with CHX, and HIF-1α protein stability was subsequently assessed. The results demonstrated that artemisinin increased the degradation rate of HIF-1α protein in both HCT-116 and LoVo cells (Figure 3A). To further elucidate the mechanism underlying HIF-1α degradation, cells were treated with the proteasome inhibitor MG132. Artemisinin significantly reduced HIF-1α protein levels; however, in the presence of both artemisinin and MG132, this reduction was not observed. These findings indicated that artemisinin promotes proteasome-mediated degradation of HIF-1α protein (Figure 3B). To visually verify the suppressive effect of artemisinin on intracellular HIF-1α expression in vivo, immunohistofluorescence staining was performed on xenograft tumor tissues. Representative micrographs captured under ×400 magnification exhibited markedly attenuated HIF-1α fluorescent intensity in tumor tissues harvested from artemisinin-treated mice relative to the NC control group (Figure 3C). For quantitative comparison of HIF-1α abundance across groups, immunofluorescence intensity scores were quantified and subjected to statistical analysis. The quantitative data demonstrated that artemisinin intervention significantly decreased the HIF-1α staining score versus the NC group (Figure 3D). Consistent with the foregoing western blot findings, these in situ immunofluorescence observations further corroborated that artemisinin facilitates proteasome-dependent degradation of HIF-1α protein in CRC.

Figure 3 Artemisinin promotes the degradation of HIF-1α protein. (A) The half-life of HIF-1α proteins were assessed in HCT-116 and LoVo cells treated with artemisinin. (B) HCT-116 and LoVo cells with artemisinin treatment and control cells were treated with or without MG132 (5 μM) for 12 h. *, P<0.05. (C) Representative immunofluorescence images of HIF-1α in xenograft tumor tissues from NC and artemisinin-treated groups (×400 magnification and ×400 magnification). (D) Quantitative statistical analysis of HIF-1α immunofluorescence intensity scores in xenograft tumor tissues of the two groups. *, P<0.05. HIF-1α, hypoxia-inducible factor-1α; NC, negative control.

HIF-1α increased the expression level of FAM83A-AS1

HIF-1α enhances glycolysis by translocating to the nucleus and binding to the DNA of HK2 and PKM2, thereby promoting their transcription (16). Prior studies have indicated that HIF-1α levels may be associated with FAM83A-AS1 (17). The effect of artemisinin on FAM83A-AS1 expression was assessed, and the results demonstrated that artemisinin significantly reduced the level of FAM83A-AS1 (Figure 4A,4C). Stable cell lines with upregulated HIF-1α were established in HCT-116 and LoVo cells, and increased expression of HIF-1α was associated with elevated levels of FAM83A-AS1 (Figure 4A,4C). Since HIF-1α functions as a transcription factor in the nucleus, its intracellular distribution was assessed by immunofluorescence. The results indicated that upregulation of HIF-1α increased its nuclear localization (Figure 4B,4D), whereas artemisinin treatment significantly reduced intracellular HIF-1α levels (Figure 4B,4D). The expression pattern of FAM83A-AS1 was consistent with that of HIF-1α in the nucleus. These findings indicated that FAM83A-AS1 expression was regulated by HIF-1α.

Figure 4 HIF-1α regulates the expression level of FAM83A-AS1. (A) qRT-PCR was used to verify the levels of FAM83A-AS1 by up-regulating of HIF-1α and artemisinin in HCT-116 cells. (B) Immunofluorescence assay was used to detect the distribution of HIF-1α in HCT-116 cells. (C) qRT-PCR was used to verify the levels of FAM83A-AS1 by up-regulating of HIF-1α and artemisinin in LoVo cells. (D) Immunofluorescence assay was used to detect the distribution of HIF-1α in LoVo cells. *, P<0.05. HIF-1α, hypoxia-inducible factor-1α; qRT-PCR, quantitative real-time polymerase chain reaction.

FAM83A-AS1 regulated the proliferation, glycolysis, and metastasis of CRC cells

The role of FAM83A-AS1 in CRC cell function was further investigated by modulating its expression in cells. si-FAM83A-AS1 was transfected into HCT-116 and LoVo cells, and knockdown of FAM83A-AS1 expression significantly inhibited cell proliferation and migration (Figure 5A-5C). Given the metabolic characteristics of tumor cells, glycolysis assays were performed using a Seahorse analyzer. The results demonstrated that lactate production was reduced in both cell lines following FAM83A-AS1 knockdown (Figure 5D).

Figure 5 Effect of FAM83A-AS1 on CRC cells. (A) qRT-PCR was used to verify the levels of FAM83A-AS1 by down-regulating of FAM83A-AS1. (B) The effect of FAM83A-AS1 on the viability of CRC cells by MTS. (C) The effect of FAM83A-AS1 on the metastasis of CRC cells by Transwell assays. (D) The effect of FAM83A-AS1 on aerobic glycolysis in CRC cells by ECAR. (E) Relative expression levels of mRNA determined by qRT-PCR. (F) Relative protein expression levels determined by western blotting. *, P<0.05. CRC, colorectal cancer; ECAR, extracellular acidification rate; MTS, mitochondrial transmembrane potential; qRT-PCR, quantitative real-time polymerase chain reaction.

Following the observed inhibition of cell migration and glycolysis after FAM83A-AS1 knockdown, the underlying regulatory mechanisms of FAM83A-AS1 in CRC were further investigated using PCR and Western blot analyses. The results demonstrated that knockdown of FAM83A-AS1 reduced the expression of the metastasis-related gene N-cadherin and the glycolysis-related proteins PKM2 and HIF-1α in CRC cell lines (Figure 5E). Consistent findings were observed at the protein level. Following FAM83A-AS1 knockdown, the expression levels of N-cadherin, PKM2, and HIF-1α were decreased (Figure 5F).

Clinical correlation of HIF-1α in patients with CRC

A total of 219 patients with CRC were included, and the expression of HIF-1α in tumor and adjacent non-tumor tissues was determined by IHC. The results demonstrated that the positive rate of HIF-1α expression in tumor tissues was significantly higher than that in adjacent non-tumor tissues (154/219 vs. 74/219, P<0.05), and the expression level of HIF-1α was significantly elevated in tumor tissues (Figure 6A). Patients were stratified into HIF-1α-positive and HIF-1α-negative groups based on its expression in tumor tissues, and the associations between HIF-1α expression and clinical characteristics were analyzed. The results indicated that HIF-1α expression was significantly associated with tumor node metastasis (TNM) stage, tumor (T) stage, node (N) stage, carcinoembryonic antigen (CEA) levels, recurrence, and metastasis, but not with age, sex, body mass index (BMI), tumor size, or tumor location (Table 1). Patients with positive HIF-1α expression exhibited higher TNM and T stages, an increased likelihood of lymph node metastasis and recurrent metastasis, and elevated CEA levels. Prognostic factors were subsequently assessed. The results demonstrated that TNM stage, T stage, N stage, recurrent metastasis, CEA levels, and HIF-1α expression were significant prognostic factors in patients with CRC (Table 2; Figure 6B). Patients with higher TNM and T stages, presence of lymph node metastasis, recurrent metastasis, and elevated CEA and HIF-1α levels exhibited poorer prognosis.

Figure 6 Clinical correlation of HIF-1α in patients with colorectal cancer. (A) The expression level of HIF-1α in cancer tissue and para-cancer tissues were detected by IHC. (B) Factors were analyzed that influence the prognosis of patients. *, P<0.05. CEA, carcinoembryonic antigen; HIF-1α, hypoxia-inducible factor-1α; IHC, immunohistochemistry; N, node; T, tumor.

Discussion

Artemisinin is a sesquiterpene lactone compound containing an endoperoxide bridge, extracted from Artemisia annua L. (Compositae), and is associated with relatively low toxicity (10). In the present study, the mechanism by which artemisinin regulates CRC was investigated using a network pharmacology approach. GO and KEGG enrichment analyses indicated that artemisinin was primarily involved in the regulation of the ubiquitin pathway and DNA binding-related pathways, with key regulatory networks centered on HIF-1α and cyclin D1/CDK4 identified. HIF-1α induces the expression of glycolytic enzyme genes and enhances anaerobic glycolytic metabolism by binding to DNA regulatory elements of target genes, thereby supporting tumor cell survival under hypoxic conditions (18). Prior studies have demonstrated that HIF-1α can regulate PKM2, and that PKM2 gene transcription can be activated by HIF-1 (16,19). PKM2 directly interacts with the HIF-1α subunit and promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding to hypoxia-responsive elements and facilitating the recruitment of p300 (20). Through network pharmacology analysis, artemisinin was identified as targeting 96 CRC-related proteins. Among these, artemisinin may form hydrogen bonds with HIF-1α protein, thereby promoting protein degradation, reducing HIF-1α expression levels in CRC cells, and subsequently regulating PKM2 expression and inhibiting glycolysis in CRC cells. Glycolysis assays further demonstrated that lactate production was reduced following artemisinin treatment. These findings indicated that artemisinin inhibited the energy supply of CRC cells, thereby suppressing cell cycle progression and cell migration. Artemisinin may form hydrogen bonds with Cyclin D1 and CDK4, resulting in modulation of the expression of these proteins, induction of cell cycle arrest, and an increase in the proportion of CRC cells in the G1 phase from 55% to 61%. The G1 phase represents the early stage of DNA synthesis, during which RNA and ribosome synthesis predominates and requires substantial energy and biosynthetic resources. Consistent with these findings, inhibition of glycolysis by artemisinin reduced energy availability, thereby suppressing cell cycle progression and proliferation of CRC cells through dual mechanisms.

Long noncoding RNAs (lncRNAs) are defined as noncoding RNA molecules exceeding 200 bp in length (17). In general, lncRNAs regulate gene expression at multiple levels, including chromatin modification, transcription, and post-transcriptional processes. Prior studies have demonstrated that lncRNAs are involved in the regulation of the cell cycle, cell differentiation, cell migration, energy metabolism, and mRNA stabilization (21,22). LncRNAs are distributed in both the nucleus and cytoplasm, with the majority localized in the nucleus, and they exhibit tissue-specific expression patterns.

These characteristics indicate their potential utility as prognostic biomarkers. FAM83A-AS1 is transcribed from the antisense strand of the FAM83A gene located at 8q24.13. Prior studies have demonstrated that FAM83A-AS1 regulates glycolysis in lung cancer cells and promotes tumor progression (PMID: 36960858). HIF-1α is rapidly degraded in oxygenated cells via the ubiquitin–proteasome pathway (PMID: 9653127). A study by Chen et al. demonstrated that FAM83A-AS1 enhances glycolysis in lung cancer cells by inhibiting the degradation of HIF-1α (17). Preliminary experimental findings indicated that FAM83A-AS1 may be involved in the regulation of aerobic glycolysis in CRC. FAM83A-AS1 increases HIF-1α levels and regulates its translocation from the cytoplasm to the nucleus. HIF-1α functions as a key transcription factor involved in the regulation of multiple genes (PMID: 39024501). The results demonstrated that HIF-1α increased the expression of FAM83A-AS1. These findings indicated that elevated nuclear levels of HIF-1α enhanced FAM83A-AS1 expression, whereas increased expression of FAM83A-AS1 inhibited the degradation of HIF-1α and further elevated its levels. The FAM83A-AS1/HIF-1α axis may represent a molecular mechanism underlying the progression of CRC. Artemisinin promotes the degradation of HIF-1α and reduces the expression level of FAM83A-AS1, thereby inhibiting the progression of CRC. Of course, this conclusion still has limitations, and future studies incorporating MG132 treatment and ubiquitination assays are required to elucidate the specific molecular mechanisms.

Limitations

Several limitations of this study should be acknowledged. Although the inhibitory effect of artemisinin on tumor growth was demonstrated to some extent in vivo, the predicted interactions between artemisinin and its targets, as identified through network pharmacology, were primarily based on computational inference and therefore may require further experimental validation. While our data support a proteasome-dependent degradation of HIF-1α induced by artemisinin, the precise molecular mechanism remains to be fully elucidated. Similarly, the exact RNA motif of FAM83A-AS1 and the protein domain of HIF-1α responsible for their physical interaction have not been mapped. The in vitro experiments were conducted in only two CRC cell lines (HCT-116 and LoVo). In addition, the generalizability of our findings to other molecular subtypes of CRC (e.g., KRAS-mutant, BRAF-mutant, or microsatellite instability-high tumors) remains to be determined using a broader panel of cell lines, organoid models, or genetically engineered mouse models.

Conclusions

In summary, our network pharmacology prediction combined with cellular and animal experiments verified that artemisinin exerts multi-faceted anti-tumor effects in CRC, including suppressing cell proliferation, G1-phase cell cycle arrest, inhibiting migration and aerobic glycolysis. Mechanistically, artemisinin facilitates proteasome-dependent degradation of HIF-1α protein, thereby breaking the positive feedback loop between HIF-1α and lncRNA FAM83A-AS1. Clinical tissue data from 219 CRC patients demonstrated that elevated HIF-1α expression in tumor lesions correlates with advanced TNM stage, lymph node metastasis, higher serum CEA, postoperative recurrence and unfavorable overall survival, further supporting the clinical value of targeting HIF-1α. Collectively, this study elucidates a novel anti-CRC mechanism of artemisinin relying on the FAM83A-AS1/HIF-1α regulatory axis, and provides preclinical evidence for repurposing artemisinin as an auxiliary therapeutic agent for CRC in surgical comprehensive treatment.

Acknowledgments

We would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0495/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0495/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0495/prf

Funding: This study was supported by the National Natural Science Foundation of China (No. 82272909).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0495/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Medical Ethics Committee of The Fourth Hospital of Hebei Medical University (No. 2022KY112). The informed consent for the use of the patients’ samples (tissue/blood samples) in this study has been obtained. Animal experiments were performed under a project license (No. 2022065) granted by the Animal Welfare Ethics Committee of The Fourth Hospital of Hebei Medical University, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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