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. 2025 Oct 6;41(1):135. doi: 10.1007/s10565-025-10084-w

Targeting Skp2 by Tanshinone IIA overcomes chemoresistance in colorectal cancer

Xin Dong 1,#, Kexin Li 1,#, Ruirui Wang 1,2,#, Baojun Wei 1, Yiling Li 1, Yu Zhang 1, Shengkai Huang 1, Guojing Wang 1, Quanquan Gao 1, Wei Li 2,✉, Wei Cui 1,✉
PMCID: PMC12500759  PMID: 41051621

Abstract

Fluorouracil (5-Fu)-based chemotherapy is a first-line treatment option for advanced colorectal cancer (CRC). However, long-term use of 5-Fu often leads to chemoresistance, which limits its therapeutic efficacy, highlighting the need for developing novel regimens to improve CRC treatment outcomes. In this study, we found that Tan IIA inhibits aerobic glycolysis in CRC cells via suppressing Skp2/Akt/HK2 signaling axis and thereby overcomes 5-Fu resistance. Specifically, Tan IIA induces ubiquitination-mediated Skp2 degradation by attenuating the interaction between USP2 and Skp2. Moreover, the combination of Tan IIA with USP2 inhibitor ML364 overcomes 5-Fu resistance in vitro and xenograft mouse models. This study elucidates a novel mechanism of 5-Fu resistance and offers a promising combination treatment option for overcoming chemoresistance.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10565-025-10084-w.

Keywords: Colorectal cancer, Glycolysis, Tanshinone IIA, Skp2, HK2

Introduction

Colorectal cancer (CRC), a prevalent digestive tract cancer, is the third most frequent cancer and exhibits a high mortality rate worldwide (Chen et al. 2022; Xi and Xu 2021). Several etiologies have been demonstrated to be related to the pathogenesis of CRC, encompassing genetic predispositions and environmental influences, such as obesity, smoking and alcohol intake (Bien and Lin 2021; Li et al. 2022b, 2022c; Jiang et al. 2022b, 2022c; Hull et al. 2020). The rapid development of multi-disciplines, early screening and diagnosis, and multiple treatment strategies, such as surgery, radiotherapy, immunotherapy, chemotherapy and combination therapeutic strategies have yielded some benefits for colorectal cancer patients (Wang and Pan 2022; Kavitha et al. 2022; Yang et al. 2022; Shinji et al. 2022). However, adverse effects and therapeutic resistance remain the major obstacles to clinical treatment (Deng et al. 2022; Peña et al. 2022). Exploring the potential mechanisms of treatment failure and discovering alternative treatment approaches are critically urgent.

The metabolic reprogramming, switching from oxidative phosphorylation (OXPHOS) to glycolysis to support genome replication and rapid growth even in oxygen-sufficient environments, is a primary characteristic of energy metabolism in cancer cells, which is known as the aerobic glycolysis or “Warburg effect” (Fukushi et al. 2022; Jiang et al. 2022a). The glycolysis pathway is controlled by certain metabolic enzymes, including phosphoglycerate kinase 1 (PGK1), phosphofructokinase (PFK), and hexokinase (HK), which are responsible for the abnormally activated energy metabolism of cancer cells (Lunt and Vander Heiden 2011; VanDer Heiden et al. 2009). Among them, HK, as the first step enzyme in glycolysis (Seiler et al. 2022), contains four isoforms HK1/2/3/4 in mammals. Accumulating evidence shows that HK2 is often overexpressed in the majority of human malignancies, including cervical cancer (Chen et al. 2021; Liu et al. 2022a), breast cancer (Ishfaq et al. 2022) and bladder cancer (Afonso et al. 2023; Huang et al. 2022a), while its expression remains relatively low in normal adult tissues (Ciscato et al. 2021). HK2 complexed with mitochondrial voltage-dependent anion channels (VDACs), which on the one hand overcomes the feedback inhibition of its product G-6-P (glucose-6-phosphate), resulting in faster ATP generation; and on the other hand, this complex safeguards cancer cells against apoptosis by stabilizing the mitochondrial outer membrane (MOM) (Mathupala et al. 2009). A multitude of investigations have demonstrated that HK2 overexpression is a major contributor to the tumorigenesis, progression, and therapeutic tolerance of cancer cells. Huang et al. discovered that HK2 expression was significantly associated with poor prognosis in glioma patients, and blocking HK2 expression showed a significant association with unfavorable prognosis in glioma cells (Huang et al. 2022b). In lung adenocarcinoma tissues, Akt/HK2 signaling activation induced by TRIM46 contributed to increasing the proliferation and cisplatin resistance of cancer cells (Tantai et al. 2022). Based on the critical pro-tumor effect of HK2, the exploration of drugs targeting HK2 or glycolysis has been the focus of tumor treatment in recent decades.

Natural products have become an increasingly popular alternative for cancer treatment due to their extensive sources, lower toxicity, and high bioactivities (Zhu et al. 2022). Tanshinone IIA (Tan IIA) is a major pharmacologically lipophilic component derived from the traditional Chinese medicinal herb Salvia miltiorrhiza Bunge (Danshen) (Li et al. 2008). Modern pharmacological research indicates that Tan IIA has extensive miscellaneous biological activities such as antioxidant, antiangiogenic, anti-atherosclerotic, anti-myocardial ischemia, and anti-inflammatory activities (Ansari et al. 2021; Li et al. 2020). Importantly, increasing evidence has reported that Tan IIA has a wide range of antitumor activities in various human cancer cell lines (Fang et al. 2020). For example, it can promote ferroptosis in cutaneous melanoma via the STAT1/PTGS2 axis (Chen et al. 2025). In colorectal cancer cells, Tan IIA upregulated the expression of miR-30b-5p to promote the sensitivity of CRC cells to oxaliplatin (Ge and Zhang 2022). In this present study, the effect of Tan IIA on glucose metabolism in CRC cells was first detected. Tan IIA was identified to significantly inhibit CRC growth and reverse 5-Fu resistance in vitro and in vivo, and the potential mechanism of its antitumor effect in CRC cells was investigated.

Materials and methods

Reagents and antibodies

The chemicals reagents, including Tanshinone IIA, SZL P1-41, MK2206, cycloheximide (CHX), MG132, and ML364 were purchased from Selleck Chemicals, and DMSO, SDS, NaCl and Tris base were purchased from Sigma-Aldrich. Cell culture media and fetal bovine serum were products from Invitrogen. Lipofectamine™ 2000 was obtained from Thermo Fisher Scientific. Antibodies against HK2 (#2867, immunoblotting (IB): 1:2000), HK1 (#2024, IB: 1:2000), cleaved-caspase 3 (#9661, IB: 1:1000), Bax (#14796, IB: 1:1000), VDAC1 (#4866, IB:1:2000), α-Tubulin (#2144, IB:1:10000), β-actin (#3700, IB: 1:10,000), cytochrome C (#11940, IB: 1:1000), Akt (#4691, IB: 1:2000), p-Akt (#4060, IB: 1:1000), Skp2 (#2652, IB:1:2000, IHC: 1:100), anti-mouse IgG HRP (#7076), and anti-rabbit IgG HRP (#7074) were purchased from Cell Signaling Technology. Anti-Ki67 antibody (ab16667, IHC: 1: 250) and donkey anti-rabbit IgG H&L (Alexa Fluor® 568, ab175470) were acquired from Abcam.

Cell lines and cell culture

Normal human colonic epithelial cells FHC and colorectal cancer cells HCT116, HT29, and SW620 were acquired from ATCC. All cells were cultured in a humidified incubator (37 °C, 5% CO2). The 5-Fu-resistant cell lines HCT116R and HT29R were generated in our laboratory via continuous exposure of parental cells to gradually escalating 5-Fu concentrations over ~ 6 months (Gan et al. 2023).

MTS assay

The viability of CRC cells was assessed using an MTS assay. Specifically, HCT116 and HT29 cells (2 × 103 cells/well) were plated in 96-well plates and subjected to treatment with varying concentrations of Tan IIA or left untreated. Finally, each well added the MTS regent (#G3581, Promega) and incubated for an additional hour.

Anchorage-independent cell growth assay

CRC cells were either exposed to varying concentrations of Tan IIA or left untreated, then counted and resuspended at a density of 8 × 103 cells/well in 1 ml of 0.3% agar supplemented with 10% FBS Eagle’s medium. This cell suspension was plated onto a 6-well plate pre-coated with a 0.6% agar base, followed by incubation at 37 °C in a 5% CO2 incubator for two weeks. Subsequently, the number of colonies was enumerated under a microscope.

Glycolysis analysis

CRC cells were exposed to DMSO (as a control) or escalating concentrations of Tan IIA for 24 h. Cells were then seeded into 6-well plates at a density of 1 × 10⁶ per well and incubated for 6 h. After that, cells were cultured in fresh medium for an additional 8 h. Glycolysis analyses were performed at the Laboratory of Hunan Cancer Hospital of Central South University. The relative rates of glucose consumption and lactate production were normalized against the protein concentration of the samples.

Western blotting (WB)

Protein preparation and IB were conducted as previously reported (Liu et al. 2019). Briefly, cells were lysed in RIPA buffer containing protease inhibitors and kept on ice for 30 min. Protein concentrations in the resulting lysates were then quantified using the BCA Assay Reagent (#23228, Thermo Fisher Science). Protein samples (20 μg) were separated via SDS-PAGE electrophoresis and then transferred onto a PVDF membrane, followed by incubation with 5% skim milk for 1 ~ 2 h. Primary antibodies were applied to the membrane and incubated overnight at 4 °C, and secondary antibody was added and incubated for 30 min at room temperature. The protein bands were then visualized using ECL reagents.

Immunofluorescence (IF) staining

CRC cells were treated with a DMSO (as a control) or escalating concentrations of Tan IIA for 24 h, and then fixed in ice-cold 4% paraformaldehyde, followed by permeabilized with 0.5% Triton X-100 for 30 min. Afterward, the cells were blocked using 10% goat serum albumin dissolved in PBS for 1 h, prior to incubation with the primary antibody overnight at 4 °C. Subsequently, the secondary antibody–Alexa Fluor 568 dye-labeled anti-rabbit IgG was incubated for 40 min. Nuclear staining was performed using DAPI. Images were visualized and captured using a confocal fluorescence microscopy system (NIKON C1si; NIKON Instruments Co.).

Immunohistochemical (IHC) staining

Mice xenograft tumor tissue slides were dewaxed and rehydrated through a graded alcohol series, followed by soaking in boiling 10 mM sodium citrate buffer (pH 6.0) for 10 min to perform antigen retrieval. Endogenous peroxidase activity was blocked with 3% H2O2 (10 min). Subsequently, blocking buffer containing 50% goat serum albumin was applied to the sections, which were then incubated for 1 h. After blocking, the tissues were incubated with primary antibodies overnight at 4 °C, and this was followed by incubation with secondary antibody for 45 min. Counterstaining was performed using hematoxylin. Finally, the sections were imaged under a light microscope, and the images were analyzed using Image Pro Plus software.

In vivo tumor growth

The xenograft models were generated with HCT116 (1 × 106) or HT29 (1 × 106) cells injected into the right flank of 6-week-old athymic nude mice (n = 4 per group). Once tumor volumes reached 100 mm3, mice in the compound-treated group received intraperitoneal injections of Tan IIA at 5 mg/kg every 2 days, while the control group was given an equivalent volume of vehicle. For combination treatment, tumor-bearing mice were randomly assigned to four groups (n = 5 per group): 1, vehicle control (0.5% dimethyl sulfoxide, 100 µL/every 2 days, i.p.); 2, ML364 (5 mg/kg/every 2 days, i.p.); 3, Tan IIA (5 mg/kg/every 2 days, i.p.); 4, ML364 (5 mg/kg/every 2 days, i.p.) + Tan IIA (5 mg/kg/every 2 days, i.p.). Tumor volume and mouse body weight were measured every 2 days. Tumor volume was calculated using the formula: length × width × width/2. At the endpoint, mice were euthanized, and the xenografts were dissected, weighed, and processed for IHC staining.

Statistical analysis

Statistical analyses were performed using SPSS (version 16.0) and GraphPad Prism 5 (GraphPad 5.0). Comparisons between two groups were analyzed using Student’s t-test, while differences among three or more groups were evaluated by one-way analysis of variance (ANOVA). All quantitative data are presented as the mean ± SD from three independent experiments.

Results

Tan IIA inhibits CRC cells

To test the inhibitory effect of Tan IIA (Fig. 1A), we treated human non-tumor colonic epithelial cells FHC (Fig. 1B) and CRC (HCT116, HT29, SW620) (Fig. 1C) cells with Tan IIA for different times, respectively. The results demonstrated that the cell viability of FHC was not markedly affected even at the highest concentration (5 μM) treated for 72 h, while the survival rate of each CRC subject group under the same conditions was decreased by more than 70%. These results suggest that Tan IIA is less toxic to immortalized non-tumor cells but inhibits CRC growth in a dose- and time-dependent manner. We inoculated CRC cells in soft agar, and the analysis suggests that Tan IIA impaired the colony formation potential of all tested CRC cells (Fig. 1D). IF analysis showed that Tan IIA inhibited CRC proliferation in a dose-dependent manner, as the Ki67-positive cells were reduced significantly (Fig. 1E). Collectively, these findings demonstrate that Tan IIA selectively impairs the tumorigenic potential of CRC cells.

Fig. 1.

Fig. 1

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Tan IIA reduces the tumorigenic properties of CRC cells. A, The chemical structure of Tan IIA. B, Immortalized FHC non-tumor cells were treated with Tan IIA (1, 2, 5 μM) or DMSO, and cell viability was examined by MTS assay. C, HCT116 (left), HT29 (middle), and SW620 (right) cells were treated with the Tan IIA (1, 2, 5 μM) or DMSO. Cell viability was examined by MTS assay. *p < 0.05, **p < 0.01, ***p < 0.001. D, The soft agar assay analysis of colony formation of the HCT116, HT29, and SW620 cells with Tan IIA (1, 2, 5 μM) or DMSO treatment. Left, representative images. Right, Quantification. ***p < 0.001. Scale bar, 200 μm. E, IF analysis of the Ki67 in Tan IIA (1, 2, 5 μM) or DMSO-treated HCT116 cells. ***p < 0.001. Scale bar, 25 μm. Comparisons were performed by using one-way ANOVA test (B-E)

Tan IIA inhibits glycolysis in CRC cells via downregulation of HK2

During the experiment, we found that following Tan IIA treatment, the yellowing of the CRC cell culture medium occurred at a significantly slower rate compared to the control group (data not shown). We speculate this phenotype is due to the inhibitory effect of Tan IIA on CRC glycolysis. Glucose consumption (Fig. 2A) and lactate production (Fig. 2B) were reduced to varying degrees in the Tan IIA-treated group, up to 70% compared to the DMSO-treated cells. IB results revealed that Tan IIA dose-dependently downregulated the protein levels of HK2. At the same time, HK1 was unaffected (Fig. 2C). This suggests that Tan IIA has the ability to suppress aerobic glycolysis. Overexpression of HK2 has been shown to enhance the survival of cancer cells through sustaining ATP production and protecting cancer cells from apoptotic cell death (Swargiary and Mani 2021). We observed that the protein level of cleaved-caspase 3 was seemingly increased in CRC cells following Tan IIA treatment (Fig. 2D). Notably, WB analysis revealed that with increasing Tan IIA concentration, we observed increased accumulation of cytochrome C in the cytosolic fraction and a corresponding decrease in the mitochondrial fraction. Translocation of BAX from the cytoplasm to the mitochondria also showed a sustained increase after Tan IIA treatment (Fig. 2E). For determining whether HK2 is not required for Tan IIA-induced apoptosis, we artificially overexpressed HK2 in CRC cells. The results showed that HK2 overexpression reduced Tan IIA-induced cleaved-caspase 3 expression (Fig. 2F) and rescued glucose consumption (Fig. 2G) and lactate production (Fig. 2H) in HT29 after Tan IIA treatment. We checked HK2 mRNA levels and found that Tan IIA inhibits HK2 transcription (Fig. 2I). To further clarify the regulatory mechanism of HK2 downregulation, we performed CHX chase assays, which showed that Tan IIA treatment did not significantly shorten the half-life of HK2 protein (Fig. S1), suggesting that post-translational degradation is not the primary cause of HK2 reduction. In conclusion, these results suggest that Tan IIA triggers intrinsic apoptosis in CRC cells in an HK2-dependent way.

Fig. 2.

Fig. 2

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Tan IIA inhibits glycolysis in CRC cells through the downregulation of HK2. A, Glucose consumption in HCT116 and HT29 cells treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h. *p < 0.05, ***p < 0.001. B, Lactate production in HCT116 and HT29 cells treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h. **p < 0.01, ***p < 0.001. C, HCT116, and HT29 cells were treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h, and whole-cell extract (WCE) was subjected to IB analysis. D, HCT116, and HT29 cells were treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h, and WCE was subjected to IB analysis. E, HT29 cell was treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h, and subcellular fractions were isolated and subjected to IB analysis. F, HT29 cells were transfected with a Flag-HK2 construct for 24 h and treated with Tan IIA (5 μM) for an additional 24 h, WCE was subjected to IB analysis. G and H, HT29 cells were transfected with a Flag-HK2 construct for 24 h and treated with Tan IIA (5 μM) for an additional 24 h, the cell culture medium was subject to glucose consumption and lactate production analysis. ***p < 0.001. I, HT29 cells were treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h, the mRNA levels of HK2 were examined by qRT-PCR. **p < 0.01, ***p < 0.001. Comparisons were performed by using one-way ANOVA test (A, B and G-I)

The inhibitory effect of Tan IIA on glycolysis requires targeting Skp2

Next, we investigated the signaling pathway through which Tan IIA regulates glycolysis in CRC cells. Interestingly, the results showed that Tan IIA reduced Skp2 protein expression dose-dependently (Fig. 3A). Given that Skp2 functions as an E3 ligase to promote Akt K63-linked ubiquitination and activation, we further performed ubiquitination analysis (Paccosi et al. 2023). Ubiquitination analysis showed that Tan IIA suppressed Akt Ub-k63 linked poly ubiquitination and downregulated p-Akt and HK2 (Fig. 3B). This finding aligns with earlier research demonstrating that Skp2 inhibition reduces Akt activity and glycolysis (Chan et al. 2012). Tan IIA-decreased p-Akt, HK2, and glycolysis were rescued after overexpression of Skp2 (Fig. 3C and D). The specific Skp2 inhibitor SZL P1-41 was used to mimic Skp2 deficiency (Li et al. 2022a). As expected, p-Akt, as well as HK2 protein levels in CRC treated with SZL P1-41, were decreased dose-dependently (Fig. 3E), and the efficacy of both glucose uptake (Fig. 3F) and lactate production (Fig. 3G) was remarkably diminished, suggesting that Skp2 is essential for the aerobic glycolysis in CRC cells. Functioning as a highly selective inhibitor targeting Akt1/2/3, MK-2206 (Emdal et al. 2022) i reduced Akt activity, downregulated HK2 expression (Fig. 3H), and decreased glucose consumption as well as lactate production (Fig. 3I). The IB data revealed that ectopic overexpression of constitutively activated Akt1 reversed the Tan IIA-induced reductions in Akt phosphorylation, HK2 expression, and glycolysis, even when Tan IIA was present (Fig. 3J and K). Our data suggest that the inhibition of CRC glycolysis by Tan IIA partially depends on suppressing the Skp2-Akt-HK2 axis.

Fig. 3.

Fig. 3

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Tan IIA inhibits Skp2 signaling. A, HCT116 and HT29 cells were treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h, WCE was subjected to IB analysis. B, HT29 cells were treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h, WCE was subjected to ubiquitination assay. C and D, HT29 cells were transfected with Skp2 or vector control and treated with Tan IIA (5 μM) or DMSO for 24 h, WCE was subjected to IB analysis (C), cell culture medium was subjected to glycolysis analysis (D). ***p < 0.001. E, HCT116 and HT29 cells were treated with SZL P1-41 (5, 10, 20 μM) or DMSO for 24 h, WCE was subjected to IB analysis. F and G, HCT116 and HT29 cells were treated with SZL P1-41 (5, 10, 20 μM) or DMSO for 24 h, then the cell culture medium was subject to glycolysis analysis. *p < 0.05, **p < 0.01, ***p < 0.001. H, HT29 cells were treated with MK2206 (1, 2, 4 μM) or DMSO for 24 h, WCE was subjected to IB analysis. I, HT29 cells were treated with MK2206 (1, 2, 4 μM) or DMSO for 24 h, the cell culture medium was subject to glucose consumption (left) and lactate production (right) analysis. *p < 0.05, **p < 0.01, ***p < 0.001. J, HT29 cells were transfected with active-Akt (Myr-Akt1) or vector control for 24 h, and treated with Tan IIA (5 μM) or DMSO for 24 h, WCE was subjected to IB analysis. K, HT29 cells were transfected with active-Akt (Myr-Akt1) or vector control for 24 h, and treated with Tan IIA (5 μM) or DMSO for 24 h, the cell culture medium was subject to glucose consumption (left) and lactate production (right) analysis. ***p < 0.001. Comparisons were performed by using one-way ANOVA test (D, G, F, I and K)

USP2 rescues Skp2 degradation in Tan IIA-treated CRC cells

To clarify the mechanism underlying Tan IIA-mediated Skp2 downregulation, we treated HCT116 and HT29 cells with various concentrations of Tan IIA. The IB results revealed that both Skp2 and p-Skp2 (S64) levels were lower in Tan IIA-treated CRC cells (Fig. 4A). Ser64 phosphorylation is crucial for maintaining Skp2 stability, and thus the reduction in p-Skp2 (S64) may contribute to Skp2 downregulation (Rodier et al. 2008; Geng et al. 2017). Furthermore, administration of the proteasome inhibitor MG132 reinstated Skp2 downregulation (Fig. 4B). When CHX was present, Tan IIA shortened the half-life of endogenous Skp2 from 2 h to less than 1 h (Fig. 4C). This all indicates that Tan IIA destabilized Skp2. The endogenous ubiquitination results further suggest that Tan IIA-induced ubiquitination of Skp2 dose-dependently (Fig. 4D). USP2 stabilizes Skp2 by deubiquitination (Zhang et al. 2021). In the present study, we used Co-IP to verify the possibility that Tan IIA regulates Skp2 via USP2 (Fig. 4E; Fig. S2A and S2B). The results showed that USP2 binds with Skp2 in CRC cells, and this interaction was attenuated by Tan IIA induction. We transiently transfected USP2 into HT29 cells and performed IB analysis. The result showed that ectopic overexpression of USP2 restored Skp2 and HK2 protein levels, and rescued the glycolytic inhibition in CRC cells in response to Tan IIA treatment (Fig. 4F and G). To further confirm whether USP2 is involved in Tan IIA-induced Skp2 ubiquitination, we examined the effect of USP2 overexpression on Skp2 ubiquitination in Tan IIA-treated cells. As shown in Supplementary Fig. 2C, ectopic overexpression of USP2 significantly reversed the increase in Skp2 ubiquitination induced by Tan IIA, which directly supports that USP2 counteracts Tan IIA-mediated Skp2 ubiquitination (Fig. S2C). Conversely, shRNA-mediated knockdown of USP2 led to reduced Skp2 and HK2 protein levels (Fig. S2D) and exacerbated glycolytic inhibition (Fig. S2E-F). Collectively, these gain- and loss-of-function data demonstrate that USP2 rescues Skp2 from Tan IIA-induced degradation, underscoring the critical role of USP2 in antagonizing Tan IIA-mediated regulation of Skp2 stability.

Fig. 4.

Fig. 4

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Tan IIA inhibits USP2-mediated Skp2 deubiquitination. A, HCT116 and HT29 cells were treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h, WCE was subjected to IB analysis. B, HCT116 and HT29 cells were treated with Tan IIA (5 μM) or DMSO for 24 h, followed by 20 μM MG132 treatment for 6 h, WCE was subjected to IB analysis. C, HT29 cells were treated with Tan IIA (5 μM) or DMSO for 24 h, followed by CHX treated for various time points, WCE was collected and subjected to IB analysis. D, HT29 cells were treated with Tan IIA (1, 2, 5 μM) or DMSO for 24 h, MG132 was added to the cell culture medium and maintained for 6 h. WCE were subjected to ubiquitination assay. E, HT29 cells were treated with Tan IIA (5 μM) or DMSO for 24 h, MG132 was added to the cell culture medium and maintained for 6 h. WCE was subjected to Co-IP assay. F and G, HT29 cells were transfected with Flag-USP2 construct for 24 h and treated with Tan IIA (5 μM) for an additional 24 h. WCE was collected and subjected to IB analysis (F), the cell culture medium was subject to glucose consumption (left) and lactate production (right) analysis (G). ***p < 0.001. Comparisons were performed by using one-way ANOVA test (G)

Tan IIA inhibits tumor development in CRC cells in vivo

To identify the in vivo antitumor activity of Tan IIA, we conducted xenograft mice models using HCT116 and HT29 cells. The findings displayed that the tumor volume in the vehicle control group was 823 ± 157 mm3, while that in the Tan IIA-treated group was 354 ± 73 mm3. In addition, the Tan IIA-treated group significantly reduced tumor mass and weight (Fig. 5A-C). Similarly, Tan IIA had a comparable inhibitory effect on HT29-derived xenograft tumors (Fig. 5D-F). The average weight gain of mice throughout the study suggests that Tan IIA is a well tolerate antitumor agent (Fig. 5G). IHC analysis was conducted to evaluate the protein levels of Ki67, Skp2 and HK2 in HCT116 xenograft tumors (Fig. 5H). The results showed a marked decrease in Ki67 expression levels in Tan IIA-treated tumors, consistent with the suppressed tumor growth. Moreover, Tan IIA administration led to a significant reduction in Skp2 protein expression, accompanied by a significant downregulation of HK2 levels in tumor tissues. These in vivo findings of reduced Skp2 and HK2 are in line with our in vitro observations, further supporting the involvement of the Skp2/Akt/HK2 axis in Tan IIA-mediated antitumor effects. Overall, our findings suggest that Tan IIA exhibits promising antitumor effects in CRC xenograft models.

Fig. 5.

Fig. 5

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Tan IIA inhibits tumor development in vivo. A-C, HCT116-derived xenograft tumors were treated with vehicle control or Tan IIA for different periods, tumor volume (A), mass image (B), and weight (C) were measured separately. Scale bar, 1 cm. **p < 0.01, ***p < 0.001. D-F, HT29-derived xenograft tumors were treated with vehicle control or Tan IIA for different periods, tumor volume (D), mass image (E), and weight (F) were measured separately. Scale bar, 1 cm. **p < 0.01, ***p < 0.001. G, The body weight of tumor-bearing mice with vehicle or Tan IIA treatment. H, IHC staining (left) and qualification (right) of Ki67, Skp2 and HK2 in HT29-derived xenograft tumors with vehicle or Tan IIA treatment. Scale bar, 25 μm. ***p < 0.001. Comparisons were performed using Student’s t-test (A, C, D, F and H)

Targeting Skp2 overcomes 5-Fu resistance

To further clarify the mechanism underlying 5-Fu resistance and lay a theoretical foundation for clinical therapy, we established two 5-Fu-resistant cells, HCT116R and HT29R (Yu et al. 2020). MTS assay results demonstrated that exposure to 100 µM 5-Fu for 24 h notably decreased cell viability in the parental HCT116 and HT29 cell lines. In contrast, the cell viability of the 5-Fu-resistant HCT116R and HT29R cells was unchanged (Fig. 6A). IB results showed higher protein expression of Skp2, USP2, and HK2 in 5-Fu-resistant cells than in their respective parental cells (Fig. 6B). A marked elevation in glucose consumption and lactate production was likewise noted in 5-Fu-resistant cells (Fig. 6C). Moreover, the level of cleaved-caspase 3 protein expression in HCT116R and HT29R cells after 5-Fu treatment was lower than that in their parental cells (Fig. 6D-E). Subsequently, we knocked down Skp2 in HT29R and HCT116R (Fig. 6F) and transplanted shCtrl and shSkp2 HT29R into mouse models. Depletion of Skp2 could be observed to inhibit tumor growth. Tumor volume, size, and mass as well as the degree of tumor cell proliferation, Ki67, showed a significant reduction compared to tumors without Skp2 knockdown (Fig. 6G-J). These results suggest that knockdown of Skp2 impaired the tumorigenicity of CRC cells. In addition, the MTS results showed an increased sensitivity of cells to 5-Fu after knockdown of Skp2 (Fig. 6K), indicating that Skp2 is necessary to maintain chemoresistance in CRC cells.

Fig. 6.

Fig. 6

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Targeting Skp2 overcomes 5-FU resistance. A, MTS assay analysis of cell viability of parental and 5-Fu-resistant cells (HCT116/HCT116R and HT29/HT29R). B, IB analysis of Skp2, HK2 and USP2 protein level in 5-Fu-resistant and parental cells (HCT116/HCT116R and HT29/HT29R). C, Glucose consumption (left) and lactate production (right) of 5-Fu-resistant CRC cells and their parental cells (HCT116/HCT116R and HT29/HT29R). **p < 0.01, ***p < 0.001. D, HCT116 and HCT116R cells were treated with 5-Fu (100 μM) or DMSO for 24 h, WCE was subjected to IB analysis. E, HT29 and HT29R cells were treated with 5-Fu (100 μM) or DMSO for 24 h, WCE was subjected to IB analysis. F, IB for Skp2 expression in shCtrl and shSkp2 HT29R and HCT116R cells. G-I, Tumor volume (G), tumor mass image (H), and weight (I) of HT29R-shCtrl and HT29R-shSkp2 xenograft tumors. Scale bar, 1 cm. ***p < 0.001. J, IHC staining (left) and qualification (right) of Ki67 in HT29R-shCtrl and HT29R-shSkp2 xenograft tumors. Scale bar, 25 μm. ***p < 0.001. K, shCtrl and shSkp2 HT29R cells were treated with 5-Fu or DMSO for 24 h, cell viability was examined by MTS assay. ***p < 0.001. Comparisons were performed using Student’s t-test (A, C and K) and one-way ANOVA (G, I and J)

The combination of Tan IIA and ML364 overcomes chemoresistance in CRC

To determine whether Tan IIA affects chemotherapy in colorectal cancer cells, we co-cultured this compound and ML364 (specific USP2 inhibitor) (Davis et al. 2016) with HCT116R and HT29R cells. The rationale for this combination therapy stems from our mechanistic insights: Tan IIA disrupts the USP2-Skp2 interaction to promote Skp2 degradation, while ML364 directly inhibits USP2’s deubiquitinase activity that is critical for Skp2 stabilization. We hypothesize that their combined action may dually target the USP2-Skp2 axis, resulting in synergistic enhancement of Skp2 degradation and consequently more potent suppression of the Skp2/Akt/HK2-mediated glycolytic pathway and 5-Fu resistance. It was seen that either Tan IIA or ML364 alone reduced the cell viability of 5-Fu-resistant cells, and this inhibitory efficacy was further enhanced by over 70% with the combination treatment (Fig. 7A). In addition, glucose uptake and lactate production analysis also reduced consistently (Fig. 7B-C). To test whether Tan IIA and ML364 can also overcome 5-Fu resistance in CRC cells in vivo, we xenografted HT29R and conducted Tan IIA, ML364, or combination treatment for 2 weeks. As expected, at the endpoint of the experiment, tumor weight and volume were reduced by Tan IIA or ML364 monotherapy and was further reduced by the Tan IIA/ML364 combination (Fig. 7D-F). IHC staining showed that Tan IIA reduced Skp2 and Ki67 protein expression in xenograft tumor tissues, with this suppressive effect being more pronounced in the combined therapy group (Fig. 7G-H). These results suggest that Tan IIA inhibits tumor growth and shows the potential to overcome CRC resistance. Notably, the mice did not show significant changes in body weight throughout the study period (Fig. 7I). Histopathology also showed no significant evidence of the toxicity of Tan IIA on the function of vital organs, including liver, heart, lung, kidney, and spleen (Fig. 7J). Consistently, serum biochemical assays revealed that levels of ALT, AST, BUN, and creatinine remained within normal ranges in all treatment groups, further confirming the in vivo safety of the combination therapy (Fig. S3A-C). Consistent with our in vitro findings, these in vivo results further confirm that the USP2-Skp2 axis plays a functional role in mediating chemoresistance, as the combined targeting of this axis by Tan IIA and ML364 achieves enhanced efficacy in overcoming 5-Fu resistance.

Fig. 7.

Fig. 7

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The combination of Tan IIA and ML364 overcomes chemoresistance in CRC. A, HCT116R and HT29R cells were treated with Tan IIA (5 μM), ML364 (10 μM) or a combination for 24 h, cell viability was examined by MTS assay. ***p < 0.001. B-C, HCT116R and HT29R cells were treated with Tan IIA (5 μM), ML364 (10 μM) or a combination for 24 h, the cell culture medium was subject to glucose consumption (B) and lactate production (C) analysis. ***p < 0.001. D-F, HT29R-derived xenograft tumors were treated with vehicle control, Tan IIA, ML364 or combination, the tumor mass (D), tumor volume (E), and weight (F) were measured separately. Scale bar, 1 cm. ***p < 0.001. G-H, IHC staining (G) and qualification (H) of Ki67 and Skp2 in HT29-derived xenograft tumors with vehicle control, Tan IIA, ML364 or combination treatment. Scale bar, 25 μm. ***p < 0.001. I, The body weight of tumor-bearing mice with vehicle control, Tan IIA, ML364 or combination treatment. J, HE staining of the liver, heart, lung, kidney, and spleen from the vehicle control, Tan IIA, ML364 or combination-treated mice. Scale bar, 25 μm. Comparisons were performed by using one-way ANOVA test (A-C, E, F and H)

Discussion

HK2 is the first rate-limiting step of glycolysis and catalyzes glucose phosphorylation. In multiple human solid tumors, HK2 is highly upregulated to enhance aerobic glycolysis and affect cellular growth and survival processes (Roberts and Miyamoto 2015). The expression and activity of HK2 are dynamically modulated by multiple factors, such as FGF, TGF-β, HIF-1α, and c-MYC, that act at transcriptional, translational, and post-translational levels (Yu et al. 2021; Xu et al. 2021). In addition, a serine/threonine kinase Akt has been demonstrated to directly phosphorylate HK2 Thr473 residue contributing to regulating its localization and antiapoptotic function (Zhou et al. 2022). These findings allow the drug to target HK2 as inhibiting its activity or decreasing expression (Shan et al. 2022). Benitrobenrazide (BNBZ), a novel selective HK2 inhibitor, noticeably induced apoptosis and inhibited the glycolysis and proliferation of SW1990 cancer cells (Zheng et al. 2021). Huang’s research team showed that Sulforaphane (SFN) inhibited glycolysis and OXPHOS contributed to strongly suppressing ATP production via blocking Akt/HK2 axis in bladder cancer (Huang et al. 2022a). A recent study has revealed that HuaChanSu could restrain tumor growth and inhibit glucose metabolism in hepatocellular carcinoma cells by suppressing HK2 expression (Wu et al. 2022). Notably, metabolic vulnerabilities similar to those we identify as targets of Tan IIA have been highlighted in research on CHK1 inhibitor-induced replication and metabolic stress, emphasizing the broader relevance of targeting such metabolic pathways in overcoming cancer resistance (Acharya et al. 2024). In this research, the pharmacological actions of Tan IIA on CRC cells were examined. The findings revealed that Tan IIA exerted substantial antitumor activity against CRC cells both in vitro and in vivo, which was ascribed to its function in modulating glycolytic metabolism through the inhibition of the metabolic enzyme HK2.

S-phase kinase-associated protein 2 (Skp2) is a subunit of the Skp2-Skp1-Cullin-1-F-box protein (SCF) E3 ligase complex, which promotes the ubiquitination of its substrates and their subsequent degradation via the proteasome (Frescas and Pagano 2008). Skp2 functions as an oncogene by regulating its protein substrates (e.g., p27, p21, FOXO1, Cyclin D/E, and Akt) to participate in many critical cellular processes, including the cell cycle, differentiation, metastasis, invasion, senescence, and apoptosis (Cai et al. 2020; Gupta et al. 2022; Wu et al. 2021; Hershko 2008). Notably, Skp2 is widely overexpressed in hematological malignancies and solid tumors such as leukemia (Hodeib et al. 2022), lymphoma (Yan et al. 2019), prostate cancer (Liang et al. 2022), lung cancer (Zou et al. 2022), and colorectal cancer (Yu et al. 2022), and closely related to therapeutic resistance, disease progression, and poor prognosis. Skp2 protein level was reported to be highly upregulated in small-cell lung cancer (SCLC), and it can promote SCLC progression by inhibiting apoptosis and facilitating cell migration and invasion (Zou et al. 2022). The previous study had shown that Skp2 expression was at a higher level in patients with chronic myeloid leukemia (CML), and depletion of Skp2 enhanced the sensitivity of CML cells to tyrosine kinase inhibitors (TKI) treatment (Chen et al. 2020). Several ubiquitinates or deubiquitinates have been verified to orchestrate the Skp2 expression, such as APC/CCDH1, SCFFBXW2, USP13, USP10, and USP2 (Zhang et al. 2021). SCFFBXW2 was found to bind to Skp2 and promote its ubiquitination and degradation, thus blocking the oncogenic function of Skp2 in lung cancer cells (Xu et al. 2017). In chronic myeloid leukemia cells, USP10 could stabilize and accumulate Skp2 expression via promoting its deubiquitylation, leading to BCR-ABL activation and conferring CML cells resistant to imatinib treatment (Liao et al. 2019).

Recently, some small molecule inhibitors of Skp2 have been reported. Skp2 inhibitor C1 (SKPin C1) was confirmed could induce apoptosis and reduce the proliferation of melanoma cells by preventing p27 ubiquitination from degradation (Zhao et al. 2019). SZL P1–41 inhibited tumor stem cell characteristics and progression by selectively suppressing Skp2 E3 ligase activity (Chan et al. 2013). A novel Skp2 inhibitor, AAA-237, was discovered to arrest the cell cycle and induce apoptosis in NSCLC cells via targeting Skp2 to upregulate p21Cip1 and p27Kip1 expression (Liu et al. 2022b). In this study, we verified that the natural product Tan IIA markedly reduced the phosphorylation and total protein level of Skp2 by disrupting the interaction between Skp2 and USP2, resulting in increased Skp2 K48-linked ubiquitination and degradation. Skp2 has been characterized as an E3 ligase for driving Akt K63-linked ubiquitination and activation upon IGF-1 or EGF stimulation, which led to initiating Akt-mediated crucial cellular biological processes, including cell proliferation, apoptosis, epithelial-mesenchymal transition (EMT), and glucose metabolism (Tsai et al. 2022). Further exploration of the mechanism of Tan IIA action showed that Tan IIA significantly inhibited the proliferation and glycolysis of CRC cells by reducing Skp2 expression and suppressing Akt K63-linked ubiquitination and phosphorylation to destroy HK2 activity eventually. Furthermore, we observed that the protein level of USP2, Skp2, and HK2 was increased in 5-Fu-resistant CRC cell lines, and the combination of Tan IIA and USP2 inhibitor ML364 significantly restrained the growth of chemoresistant cells. This observation aligns with recent studies on novel fluoropyrimidine polymers (e.g., CF10) that overcome 5-Fu resistance in CRC, supporting the rationale that targeting key mediators of chemoresistance can enhance therapeutic efficacy (Okechukwu et al. 2024; Sah et al. 2023). Therefore, our findings uncovered a novel perspective on the antitumor mechanism of the compound Tan IIA and provided a potential therapeutic strategy for patients with colorectal cancer.

Conclusions

In summary, our findings indicated that one of the potential mechanisms underlying the antitumor activity of Tan IIA is the interference with Skp2 ubiquitination and degradation, which in turn inhibits the Akt/HK2 signaling pathway and disrupts glycolysis (Fig. 8). In addition, Tan IIA could induce intrinsic apoptosis synergistically with USP2 inhibitor to suppress the growth of chemoresistant CRC cells. Thus, this newly identified mechanism of Tan IIA positions it as a promising therapeutic agent for targeting CRC cells.

Fig. 8.

Fig. 8

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Schematic diagram showing that Tan IIA promotes Skp2 ubiquitination and degradation by inhibiting USP2-Skp2 interaction, thereby suppressing the Skp2/Akt/HK2 axis to interfere with aerobic glycolysis and induce intrinsic apoptosis, ultimately overcoming 5-Fu resistance in colorectal cancer

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 445 KB) (445.3KB, docx)

Acknowledgements

We thank to the National Natural Science Foundation of China (No. 82302625) and the Noncommunicable Chronic Diseases-National Science and Technology Major Project (2023ZD0509500).

Author contributions

D.X., L.K.X., W.R.R., C.W., L.W., W.B.J., conceived and designed the study. D.X., L.K.X., W.R.R., L.Y.L., H.S.K., W.G.J., Z.Y., G.Q.Q., completed the experiments. L.Y.L., H.S.K., W.G.J., Z.Y., G.Q.Q., conducted data collation and statistical analysis. D.X., L.K.X., W.R.R., completed the initial manuscript. C.W., L.W., further checked and revised the manuscript. D.X., C.W., provided funding support.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82302625) and the Noncommunicable Chronic Diseases-National Science and Technology Major Project (2023ZD0509500).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

All animal experiments were approved by the Institutional Animal Care and Use Committee (No. 202009655) of Central South University (Changsha, China).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xin Dong, Kexin Li, and Ruirui Wang contributed equally to this work.

Contributor Information

Wei Li, Email: weililx@csu.edu.cn.

Wei Cui, Email: wendycuiwei@sina.cn.

References

  1. Acharya G, Mani C, Sah N, et al. Chk1 inhibitor induced parylation by targeting parg causes excessive replication and metabolic stress and overcomes chemoresistance in ovarian cancer. Cell Death Discov. 2024;10:278. 10.1038/s41420-024-02040-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Afonso J, Gonçalves C, Costa M, et al. Glucose metabolism reprogramming in bladder cancer: Hexokinase 2 (hk2) as prognostic biomarker and target for bladder cancer therapy. Cancers (Basel). 2023. 10.3390/cancers15030982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ansari MA, Khan FB, Safdari HA, et al. Prospective therapeutic potential of tanshinone IIA: an updated overview. Pharmacol Res. 2021;164:105364. 10.1016/j.phrs.2020.105364. [DOI] [PubMed] [Google Scholar]
  4. Bien J, Lin A. A review of the diagnosis and treatment of metastatic colorectal cancer. JAMA. 2021;325:2404–5. 10.1001/jama.2021.6021. [DOI] [PubMed] [Google Scholar]
  5. Cai Z, Moten A, Peng D, et al. The skp2 pathway: a critical target for cancer therapy. Semin Cancer Biol. 2020;67:16–33. 10.1016/j.semcancer.2020.01.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chan C-H, Li C-F, Yang W-L, et al. The skp2-SCF E3 ligase regulates akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis. Cell. 2012;149:1098–111. 10.1016/j.cell.2012.02.065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chan C-H, Morrow JK, Li C-F, et al. Pharmacological inactivation of Skp2 SCF ubiquitin ligase restricts cancer stem cell traits and cancer progression. Cell. 2013;154:556–68. 10.1016/j.cell.2013.06.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen X, Huang Z, Wu W, et al. Inhibition of skp2 sensitizes chronic myeloid leukemia cells to imatinib. Cancer Manag Res. 2020;12:4777–87. 10.2147/CMAR.S253367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen Q, Li L, Liu X, et al. Hexokinases 2 promoted cell motility and distant metastasis by elevating fibronectin through akt1/p-akt1 in cervical cancer cells. Cancer Cell Int. 2021;21:600. 10.1186/s12935-021-02312-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen J, Zhu H, Yin Y, et al. Colorectal cancer: Metabolic interactions reshape the tumor microenvironment. Biochimica et Biophysica Acta (BBA). 2022;1877:188797. 10.1016/j.bbcan.2022.188797. [DOI] [PubMed] [Google Scholar]
  11. Chen S, Li P, Shi K, et al. Tanshinone IIA promotes ferroptosis in cutaneous melanoma via stat1-mediated upregulation of ptgs2 expression. Phytomedicine. 2025;141:156702. 10.1016/j.phymed.2025.156702. [DOI] [PubMed] [Google Scholar]
  12. Ciscato F, Ferrone L, Masgras I, et al. Hexokinase 2 in cancer: a prima donna playing multiple characters. Int J Mol Sci. 2021. 10.3390/ijms22094716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Davis MI, Pragani R, Fox JT, et al. Small molecule inhibition of the ubiquitin-specific protease USP2 accelerates cyclin D1 degradation and leads to cell cycle arrest in colorectal cancer and mantle cell lymphoma models. J Biol Chem. 2016;291:24628–40. 10.1074/jbc.M116.738567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Deng S, Vlatkovic T, Li M, et al. Targeting the DNA damage response and DNA repair pathways to enhance radiosensitivity in colorectal cancer. Cancers (Basel). 2022. 10.3390/cancers14194874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Emdal KB, Palacio-Escat N, Wigerup C, et al. Phosphoproteomics of primary aml patient samples reveals rationale for akt combination therapy and p53 context to overcome selinexor resistance. Cell Rep. 2022;40:111177. 10.1016/j.celrep.2022.111177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fang Z-Y, Zhang M, Liu J-N, et al. Tanshinone iia: A review of its anticancer effects. Front Pharmacol. 2020;11:611087. 10.3389/fphar.2020.611087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frescas D, Pagano M. Deregulated proteolysis by the f-box proteins Skp2 and beta-Trcp: tipping the scales of cancer. Nat Rev Cancer. 2008;8:438–49. 10.1038/nrc2396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fukushi A, Kim H-D, Chang Y-C, et al. Revisited metabolic control and reprogramming cancers by means of the Warburg effect in tumor cells. Int J Mol Sci. 2022. 10.3390/ijms231710037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gan Y, Zhou L, Wang R, et al. Curcumol reduces aerobic glycolysis and overcomes chemoresistance by inducing cdh1-mediated skp2 ubiquitination. Am J Chin Med. 2023;51:723–40. 10.1142/s0192415x23500349. [DOI] [PubMed] [Google Scholar]
  20. Ge T, Zhang Y. Tanshinone IIA reverses oxaliplatin resistance in colorectal cancer through microRNA-30b-5p/aven axis. Open Med (Wars). 2022;17:1228–40. 10.1515/med-2022-0512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Geng Q, Liu J, Gong Z, et al. Phosphorylation by mTORC1 stablizes Skp2 and regulates its oncogenic function in gastric cancer. Mol Cancer. 2017;16:83. 10.1186/s12943-017-0649-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gupta P, Zhao H, Hoang B, et al. Targeting the untargetable: Rb1-deficient tumours are vulnerable to skp2 ubiquitin ligase inhibition. Br J Cancer. 2022;127:969–75. 10.1038/s41416-022-01898-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hershko DD. Oncogenic properties and prognostic implications of the ubiquitin ligase Skp2 in cancer. Cancer. 2008;112:1415–24. 10.1002/cncr.23317. [DOI] [PubMed] [Google Scholar]
  24. Hodeib H, Abd El Hai D, Tawfik MA, et al. The impact of skp2 gene expression in chronic myeloid leukemia. Genes 2022;13. 10.3390/genes13060948 [DOI] [PMC free article] [PubMed]
  25. Huang L, He C, Zheng S, et al. Akt1/hk2 axis-mediated glucose metabolism: A novel therapeutic target of sulforaphane in bladder cancer. Mol Nutr Food Res. 2022a;66:e2100738. 10.1002/mnfr.202100738. [DOI] [PubMed] [Google Scholar]
  26. Huang Y, Ouyang F, Yang F, et al. The expression of hexokinase 2 and its hub genes are correlated with the prognosis in glioma. BMC Cancer. 2022b;22:900. 10.1186/s12885-022-10001-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hull R, Francies FZ, Oyomno M, et al. Colorectal cancer genetics, incidence and risk factors: in search for targeted therapies. Cancer Manag Res. 2020;12:9869–82. 10.2147/CMAR.S251223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ishfaq M, Bashir N, Riaz SK, et al. Expression of hk2, pkm2, and pfkm is associated with metastasis and late disease onset in breast cancer patients. Genes. 2022. 10.3390/genes13030549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jiang H, Wei H, Wang H, et al. Zeb1-induced metabolic reprogramming of glycolysis is essential for macrophage polarization in breast cancer. Cell Death Dis. 2022a;13:206. 10.1038/s41419-022-04632-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jiang X, Jiang Z, Cheng Q, et al. Cholecystectomy promotes the development of colorectal cancer by the alternation of bile acid metabolism and the gut microbiota. Front Med. 2022b;9:1000563. 10.3389/fmed.2022.1000563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kavitha MS, Gangadaran P, Jackson A, et al. Deep neural network models for colon cancer screening. Cancers (Basel). 2022. 10.3390/cancers14153707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Li M-H, Chen J-M, Peng Y, et al. Investigation of danshen and related medicinal plants in China. J Ethnopharmacol. 2008;120:419–26. 10.1016/j.jep.2008.09.013. [DOI] [PubMed] [Google Scholar]
  33. Li M, Gao F, Zhao Q, et al. Tanshinone IIA inhibits oral squamous cell carcinoma via reducing akt-c-myc signaling-mediated aerobic glycolysis. Cell Death Dis. 2020;11:381. 10.1038/s41419-020-2579-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Li P, Chen T, Kuang P, et al. Aurora-A/FOXO3A/SKP2 axis promotes tumor progression in clear cell renal cell carcinoma and dual-targeting aurora-A/SKP2 shows synthetic lethality. Cell Death Dis. 2022a;13:606. 10.1038/s41419-022-04973-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Li Y, Ye D, Zhou W, et al. Alcohol consumption and colorectal cancer risk: a mendelian randomization study. Front Genet. 2022b;13:967229. 10.3389/fgene.2022.967229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Liang H, Zhang F, Hong Y, et al. Synergistic silencing of skp2 by sirna self-assembled nanoparticles as a therapeutic strategy for advanced prostate cancer. Small. 2022;18:e2106046. 10.1002/smll.202106046. [DOI] [PubMed] [Google Scholar]
  37. Liao Y, Liu N, Xia X, et al. Usp10 modulates the skp2/bcr-abl axis via stabilizing skp2 in chronic myeloid leukemia. Cell Discov. 2019;5:24. 10.1038/s41421-019-0092-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Liu W, Li W, Liu H, et al. Xanthohumol inhibits colorectal cancer cells via downregulation of hexokinases II-mediated glycolysis. Int J Biol Sci. 2019;15:2497–508. 10.7150/ijbs.37481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Liu C, Li Y, Dong C, et al. E6e7 regulates the hk2 expression in cervical cancer via gsk3β/fto signal. Arch Biochem Biophys. 2022a;729:109389. 10.1016/j.abb.2022.109389. [DOI] [PubMed] [Google Scholar]
  40. Liu J, Zheng X, Li W, et al. Anti-tumor effects of SKP2 inhibitor aaa-237 on NSCLC by arresting cell cycle at G0/G1 phase and inducing senescence. Pharmacol Res. 2022b;181:106259. 10.1016/j.phrs.2022.106259. [DOI] [PubMed] [Google Scholar]
  41. Lunt SY, Vander Heiden MG. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu Rev Cell Dev Biol. 2011;27:441–64. 10.1146/annurev-cellbio-092910-154237. [DOI] [PubMed] [Google Scholar]
  42. Mathupala SP, Ko YH, Pedersen PL. Hexokinase-2 bound to mitochondria: Cancer’s stygian link to the “warburg effect” and a pivotal target for effective therapy. Semin Cancer Biol. 2009;19:17–24. 10.1016/j.semcancer.2008.11.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Okechukwu CC, Ma X, Sah N, et al. Enhanced therapeutic efficacy of the nanoscale fluoropyrimidine polymer CF10 in a rat colorectal cancer liver metastasis model. Cancers. 2024. 10.3390/cancers16071360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Paccosi E, Balzerano A, Proietti-De-Santis L. Interfering with the ubiquitin-mediated regulation of akt as a strategy for cancer treatment. Int J Mol Sci. 2023. 10.3390/ijms24032809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Peña M, Guzmán A, Martínez R, et al. Preventive effects of Brassicaceae family for colon cancer prevention: a focus on in vitro studies. Biomed Pharmacother. 2022;151:113145. 10.1016/j.biopha.2022.113145. [DOI] [PubMed] [Google Scholar]
  46. Roberts DJ, Miyamoto S. Hexokinase II integrates energy metabolism and cellular protection: Akting on mitochondria and torcing to autophagy. Cell Death Differ. 2015;22:248–57. 10.1038/cdd.2014.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rodier G, Coulombe P, Tanguay PL, et al. Phosphorylation of skp2 regulated by cdk2 and cdc14b protects it from degradation by apc(cdh1) in g1 phase. EMBO J. 2008;27:679–91. 10.1038/emboj.2008.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sah N, Luna P, Mani C, et al. A novel second-generation nano-fluoropyrimidine to treat metastatic colorectal cancer and overcome 5-fluorouracil resistance. Cancer Research 2023;83. 10.1158/1538-7445.Am2023-6178
  49. Seiler K, Humbert M, Minder P, et al. Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions. Cell Death Dis. 2022;13:448. 10.1038/s41419-022-04891-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Shan W, Zhou Y, Tam KY. The development of small-molecule inhibitors targeting hexokinase 2. Drug Discov Today. 2022;27:2574–85. 10.1016/j.drudis.2022.05.017. [DOI] [PubMed] [Google Scholar]
  51. Shinji S, Yamada T, Matsuda A, et al. Recent advances in the treatment of colorectal cancer: a review. J Nippon Med Sch. 2022;89:246–54. 10.1272/jnms.JNMS.2022_89-310. [DOI] [PubMed] [Google Scholar]
  52. Swargiary G, Mani S. Molecular docking and simulation studies of phytocompounds derived from Centella asiatica and Andrographis paniculata against hexokinase ii as mitocan agents. Mitochondrion. 2021;61:138–46. 10.1016/j.mito.2021.09.013. [DOI] [PubMed] [Google Scholar]
  53. Tantai J, Pan X, Chen Y, et al. Trim46 activates akt/hk2 signaling by modifying phlpp2 ubiquitylation to promote glycolysis and chemoresistance of lung cancer cells. Cell Death Dis. 2022;13:285. 10.1038/s41419-022-04727-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tsai P-J, Lai Y-H, Manne RK, et al. Akt: a key transducer in cancer. J Biomed Sci. 2022;29:76. 10.1186/s12929-022-00860-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. VanDer Heiden MG, Cantley LC, Thompson CB. Understanding the warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029–33. 10.1126/science.1160809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wang H, Pan W. Challenges of chimeric antigen receptor-T/natural killer cell therapy in the treatment of solid tumors: focus on colorectal cancer and evaluation of combination therapies. Mol Cell Biochem. 2022. 10.1007/s11010-022-04568-0. [DOI] [PubMed] [Google Scholar]
  57. Wu T, Gu X, Cui H. Emerging roles of skp2 in cancer drug resistance. Cells. 2021. 10.3390/cells10051147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wu Q, Wang SP, Sun XX, et al. Huachansu suppresses tumor growth and interferes with glucose metabolism in hepatocellular carcinoma cells by restraining hexokinase-2. Int J Biochem Cell Biol. 2022;142:106123. 10.1016/j.biocel.2021.106123. [DOI] [PubMed] [Google Scholar]
  59. Xi Y, Xu P. Global colorectal cancer burden in 2020 and projections to 2040. Transl Oncol. 2021;14:101174. 10.1016/j.tranon.2021.101174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Xu J, Zhou W, Yang F, et al. The β-trcp-fbxw2-skp2 axis regulates lung cancer cell growth with fbxw2 acting as a tumour suppressor. Nat Commun. 2017;8:14002. 10.1038/ncomms14002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Xu Z, Jia K, Wang H, et al. Mettl14-regulated pi3k/akt signaling pathway via pten affects hdac5-mediated epithelial-mesenchymal transition of renal tubular cells in diabetic kidney disease. Cell Death Dis. 2021;12:32. 10.1038/s41419-020-03312-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yan W, Yang Y, Yang W. Inhibition of skp2 activity impaired atm-mediated DNA repair and enhanced sensitivity of cisplatin-resistant mantle cell lymphoma cells. Cancer Biother Radiopharm. 2019;34:451–8. 10.1089/cbr.2019.2787. [DOI] [PubMed] [Google Scholar]
  63. Yang Z, Wu G, Zhang X, et al. Current progress and future perspectives of neoadjuvant anti-PD-1/PD-L1 therapy for colorectal cancer. Front Immunol. 2022;13:1001444. 10.3389/fimmu.2022.1001444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yu X, Li W, Liu H, et al. Ubiquitination of the DNA-damage checkpoint kinase chk1 by traf4 is required for chk1 activation. J Hematol Oncol. 2020;13:40. 10.1186/s13045-020-00869-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Yu H, Yang W, Huang J, et al. Gpr120 induces regulatory dendritic cells by inhibiting hk2-dependent glycolysis to alleviate fulminant hepatic failure. Cell Death Dis. 2021;13:1. 10.1038/s41419-021-04394-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yu X, Zhou L, Liu W, et al. Skp2 stabilizes mcl-1 and confers radioresistance in colorectal cancer. Cell Death Dis. 2022;13:249. 10.1038/s41419-022-04685-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zhang F, Zhao Y, Sun Y. Usp2 is an skp2 deubiquitylase that stabilizes both skp2 and its substrates. J Biol Chem. 2021;297:101109. 10.1016/j.jbc.2021.101109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zhao H, Pan H, Wang H, et al. Skp2 targeted inhibition suppresses human uveal melanoma progression by blocking ubiquitylation of p27. OncoTargets Ther. 2019;12:4297–308. 10.2147/OTT.S203888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zheng M, Wu C, Yang K, et al. Novel selective hexokinase 2 inhibitor benitrobenrazide blocks cancer cells growth by targeting glycolysis. Pharmacol Res. 2021;164:105367. 10.1016/j.phrs.2020.105367. [DOI] [PubMed] [Google Scholar]
  70. Zhou Y, Guo Y, Tam KY. Targeting glucose metabolism to develop anticancer treatments and therapeutic patents. Expert Opin Ther Pat. 2022;32:441–53. 10.1080/13543776.2022.2027912. [DOI] [PubMed] [Google Scholar]
  71. Zhu Y, Yue P, Dickinson CF, et al. Natural product preferentially targets redox and metabolic adaptations and aberrantly active stat3 to inhibit breast tumor growth in vivo. Cell Death Dis. 2022;13:1022. 10.1038/s41419-022-05477-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Zou J, Lin Y, Hu M, et al. N-myc transcriptionally activates skp2 to suppress p27 expression in small cell lung cancer. Pathol Res Pract. 2022;238:154083. 10.1016/j.prp.2022.154083. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Supplementary file1 (DOCX 445 KB) (445.3KB, docx)

Data Availability Statement

No datasets were generated or analysed during the current study.

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