Protosappanin B enhances the chemosensitivity of 5-fluorouracil in colon adenocarcinoma by regulating the LINC00612/microRNA-590-3p/Golgi phosphoprotein 3 axis

Background 5-fluorouracil (5-FU) is conventionally used in chemotherapy for colon adenocarcinomas. Acquired resistance of 5-FU remains a clinical challenge in colon cancer, and efforts to develop targeted agents to reduce resistance have not yielded success. Protosappanin B (PSB), the main component of Lignum Sappan extract, is known to exhibit anti-tumor effects. However, whether and how PSB could improve 5-FU resistance in colon cancer have not yet been established. In this study, we aimed to explore the effects and underlying mechanisms of PSB in 5-FU-induced chemoresistance in colon adenocarcinoma. Methods Forty-seven paired colon cancer tissue samples from patients who received 5-FU chemotherapy were collected as clinical samples. Two 5-FU resistant colon cancer cell lines were established for in vitro experiments. Reverse transcription-quantitative PCR (RT-qPCR) was performed to determine the mRNA and microRNA (miRNA) expression levels in colon adenocarcinoma tissues and cell lines. Cell Counting Kit-8 (CCK-8) and flow cytometry assays were performed to evaluate cell proliferation and apoptosis, respectively. Results LINC00612 was highly expressed in colon adenocarcinoma samples and 5-FU resistant colon cancer cells. LINC00612 knockdown enhances 5-FU chemosensitivity in 5-FU resistant cells. Notably, PSB treatment attenuated LINC00612 expression in 5-FU resistant colon adenocarcinoma cells. Moreover, PSB treatment reversed the increase in LINC00612-induced 5-FU resistance. Mechanistically, LINC00612 specifically bound to miR-590-3p, which promoted 5-FU resistance in colon adenocarcinoma cells and attenuated the inhibitory effect of LINC00612 on GOLPH3 expression. Conclusion PSB attenuates 5-FU chemoresistance in colon adenocarcinoma by regulating the LINC00612/miRNA-590-3p/GOLPH3 axis. Graphical Abstract


Introduction
The National Comprehensive Cancer Network (NCCN) guidelines recommend oxaliplatin, fluorouracil, 5-fluorouracil (5-FU), capecitabine, FOLFOX (the combinational strategy of folinic acid, 5-FU, and oxaliplatin), and other chemotherapy regimens for treating colon cancer [1], all of which comprise 5-FU or 5-FU derivatives.Therefore, 5-FU-based comprehensive chemotherapy regimens are considered the gold standard for treating intermediate and advanced colon cancer [2].However, with the widespread development of chemotherapy, 5-FU resistance has become increasingly prominent, seriously impacting the prognosis of patients and is the main cause of chemotherapy failure in patients [3,4].Hence, enhancing and reestablishing the sensitivity of tumor cells to chemotherapeutic drugs and improving clinical efficacy could be valuable in anti-tumor therapy.
Long non-coding RNA (lncRNA) is a sub-type of RNA elements that lack protein-coding capability [5,6].LncRNAs promote chemotherapeutic resistance through various pathways, such as increasing drug metabolism, enhancing drug efflux, altering the cell cycle, abnormal apoptosis, and epithelial-mesenchymal transformation [7].For instance, lncRNA

Specimen collection
Forty-seven pairs of colon adenocarcinoma tissues and matched noncancerous samples (3-5 cm from the distal part of the tumor) were acquired from patients clinically diagnosed at The Second Affiliated Hospital of Fujian Medical University.All included patients underwent three cycles of 5-FU (425 mg/m 2 administered intravenously for 6-8 h for 5 days and 4 weeks as a treatment cycle) and were assessed for disease progression using the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) [25].Written informed consent was obtained from all participants.The methodologies employed in the present study were in accordance with the criteria established by the Ethics Committee of The Second Affiliated Hospital of Fujian Medical University (approval no.2021336, Quanzhou, Fujian, China).

Establishment of 5-FU-resistant cells
Colon adenocarcinoma SW620 and LOVO cells and normal colonic mucosal epithelial cells NCM460 were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplemented with 10% inactivated fetal bovine serum (Gibco, USA), which was replaced every 2 to 3 days.Cells exhibiting 80-90% confluency were digested and used in subsequent

Cell treatment
To verify the successful establishment of resistant cells, SW620 and LOVO cells were treated with 5-FU (0, 4, 8, 16, 32, and 64 μM; Nanjing Chia-Tai Tianqing Pharmaceutical Company) along with resistant cells to establish cell viability [19].Appropriate concentrations of PSB were screened for subsequent experiments by treating 5-FU SW620 and 5-FU LOVO cells with different concentrations of PSB (Shanghai Yuanye Bio-Technology Company) to measure cell viability.

Lentiviral transfection and RNA interference [40]
Briefly, logarithmic growth 5-FU SW620 and 5-FU LOVO cells were digested and cultured in 6-well plates overnight.All transfection plasmids, including small RNA interfering against LINC00612 (si-LINC00612 1# and si-LINC00612 2#), small RNA interference negative control (si-NC), elevated LINC00612 (LINC00612), blank overexpression vector (Vector), miR-590-3p mimic, mimic nc, si-GOLPH3 1#, si-GOLPH3 2#, short hairpin RNA of LINC00612 (sh-LINC00612), and sh-NC, were purchased from Shanghai GenePharma Company (China).Lipofectamine 3000 (Invitrogen, USA) was used to transfect the corresponding plasmids into the cells according to the manufacturer's instructions.Overexpression plasmid transfection: Serum-free medium (250 μL) was used to dilute the transfection reagent Lipofectamine 3000 (12.5 μL).The overexpression plasmid (4.2 μg) and P3000 (8 μL) were added to serum-free culture medium (250 μL) and mixed and incubated at room temperature for 5 min.Finally, the solution was mixed and incubated at room temperature for 15 min.Transfection of siRNA and miR: The serum-free medium (250 μL) was used to dilute the transfection reagent Lipofectamine 3000 (12.5 μL) (solution A).The siRNA or miR (12.5 μL) was added to serum-free culture medium (250 μL) to adjust the final concentration of siRNA or miR to 50 nM.The samples were then mixed and incubated at room temperature for 5 min.The liquids were then evenly mixed and incubated at room temperature for 15 min.

Reverse transcription-quantitative PCR (RT-qPCR) [41]
Total RNA was extracted from colon adenocarcinoma clinical samples and cells using an RNA extraction kit (Beijing Tiengen Biochemical, China).cDNA was obtained using a reverse transcription kit (RIBOBIO, China).The PCR reaction was conducted according to the SYBR Premix Ex Taq kit instructions (Takara, Japan), with pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s. GAPDH was used as a housekeeping control for LINC00612 and GOLPH3.U6 was used as the housekeeping control for miR-590-3p.Data were analyzed using the 2−ΔΔCt relative expression method [8].All experiments were performed in triplicates.All primer sequences are listed in Table 1.

Cell counting kit-8 (CCK-8)
Cells in the logarithmic growth phase were collected and inoculated evenly into 96-well plates (3500 cells/well).Then, 100 μL of DMEM containing 10 μL of CCK-8 was added to each well.Two hours later, absorbance was measured at 450 nm using an enzyme-linked immunoassay.Cell viability was calculated using the CCK-8 Cell Counting Kit (Vazyme Biotech Co., Ltd, China) instructions [13].

Flow cytometry
The culture medium in the 6-well plate was collected in a flow tube and trypsin was added to each well for digestion.After discarding trypsin, 1 mL of complete culture medium was added to terminate the digestion, and the cells were resuspended and collected in a flow tube.After centrifugation, the supernatant was discarded and 300 μL of binding buffer was added to resuspend the cells according to the instructions of the Cell Apoptosis Double Staining Detection Kit (Nanjing Fcmacs Biotech Company).Subsequently, 4 μL of propidium iodide and Annexin V dye were added for 15 min in the dark.The rate of apoptosis was analyzed using flow cytometry [13].

Subcutaneous tumorigenesis in nude mice
4-week-old male BALB/c nude mice were purchased, 5 × 10 6 SW620 cells were inoculated subcutaneously into the right axilla of nude mice with a sterile insulin needle, and tumor formation was observed regularly.Nude mice were randomly divided into five groups: sh-NC, sh-LINC00612, sh-NC + 5-FU, sh-LINC00612 + 5-FU, sh-LINC00612 + 5-FU + PSB.From day 5, 2 nmol sh-nc/sh-LINC00612 was injected intratumoral every 3 days for 8 cycles.Intraperitoneal injection of 30 mg/ kg 5-FU [20] was performed every 3 days from the 6th day, for 8 cycles.From day 7 onwards, 20 mg/kg PSB [19] was injected intraperitoneally every three days for eight cycles.Graft size was measured and recorded with Vernier calipers every other week.Graft volume was calculated using the formula: 1/2 × (length × width 2 ).After 28 days of feeding, the nude mice were sacrificed by cervical dislocation and the tumors were removed and weighed.

Statistical analysis
Data analysis was performed using GraphPad Prism (v8.3, GraphPad Software, Inc., La Jolla, CA, USA).Each independent experiment was conducted in triplicates.Data are expressed as mean ± standard deviation.The Student's t-test was used to compare two groups, and one-way ANOVA was used to compare multiple groups.Statistical significance was set at a p < 0.05.

LINC00612 is related to 5-FU resistance in colon adenocarcinoma
We first examined 5-FU-induced cytotoxicity in the SW620 and LOVO cell lines using the CCK-8 assay.The half-maximal inhibitory concentrations (IC50) values were 32 and 16 μM in SW620 and LOVO cells, respectively (Fig. 1A).Additionally, 5-FU resistance was characterized in 5-FU SW620 and 5-FU LOVO cells.Following treatment with increasing 5-FU doses, the IC50 values of SW620 and LOVO cells were less than 32 μM, whereas those of 5-FU SW620 and 5-FU LOVO cells were approximately 64 μM, which was approximately twofold higher than that of the parent cell line (Fig. 1B, C).Next, we selected and explored several newly discovered lncRNAs in tumors and detected their expression levels in clinical samples.The expression level of LINC00612 was significantly upregulated in colon adenocarcinoma tissues (Fig. 1D-H).Furthermore, LINC00612 expression was aberrantly elevated in colon cancer cells compared to that in NCM460 cells.Meanwhile, LINC00612 expression was further elevated in 5-FU resistant SW620 and 5-FU resistant LOVO cells compared with the untreated SW620 and LOVO cells (Fig. 1I).

LINC00612 knockdown promotes sensitivity of 5-FU chemotherapy in 5-FU SW620 and 5-FU LOVO cells
To examine the effect of LINC00612 on 5-FU induced chemoresistance in 5-FU SW620 and 5-FU LOVO cells, we transfected cells with si-LINC00612 to silence LINC00612 expression (Fig. 2A).In 5-FU SW620 and 5-FU LOVO cells, we observed that cell viability was reduced by about half after 32 μM of 5-FU treatment in the si-LINC00612 group.Meanwhile, inhibition of LINC00612 markedly suppressed cell viability compared with the si-NC group (Fig. 2B, C).

PSB treatment attenuates colon cancer cell growth by suppressing LINC00612 expression
Subsequently, the NCM460, 5-FU SW620, and 5-FU LOVO cells were treated with PSB.PSB significantly inhibited 5-FU SW620 and 5-FU LOVO cell growth in a dose-dependent manner, and even at a high concentration of 200 μg/mL, PSB was less toxic to normal cells (Fig. 3A).Furthermore, 100 μg/mL PSB, which reduced the number of 5-FU SW620 and 5-FU LOVO cells by 50%, was selected as the optimal concentration.PSB treatment decreased LINC00612 expression, as evaluated by RT-qPCR in 5-FU SW620 and 5-FU LOVO cells (Fig. 3B).To confirm whether the PSB-induced decline in LINC00612 expression could affect the functions of 5-FU SW620 and 5-FU LOVO cells, we established LINC00612 overexpressing

PSB promotes chemosensitivity of 5-FU in vivo
A nude mouse model of subcutaneous tumorigenesis was established to investigate the relationship between PSB and sensitivity to chemotherapy (Fig. 7A).In the subcutaneous tumorigenesis experiment of SW620 cells, compared with the sh-NC group, the weight (Fig. 7B) and volume (Fig. 7C) of tumor tissue in the sh-LINC00612 group was significantly reduced, which indicated that the malignant process of the tumor was inhibited after inhibiting the expression of LINC00612.The combination of sh-LINC00612 and 5-FU had a stronger anti-tumor effect than sh-LINC00612 or 5-FU alone, and PSB treatment further inhibited tumor growth and enhanced the chemotherapy sensitivity of 5-FU.To evaluate the acute toxicity of PSB in vivo, the activity of alanine transaminase (ALT), aspartate transaminase (AST), serum creatinine (Scr), and blood urea nitrogen (BUN) were measured.The levels of AST, ALT, BUN, and Scr were up-regulated significantly after 5-FU treatment, while the expression in serum with a reversing trend to normal induced by PSB treatment compared with those in the control group (Fig. 7D-G).The results suggested that PSB could alleviate the liver and kidney dysfunction in tumor xenograft mice.

Discussion
In the present study, PSB suppressed cell viability and accelerated the apoptosis of 5-FU-resistant colon adenocarcinoma cells by regulating the LINC00612/miR-590-3p/GOLPH3 axis (Graphical abstract).Currently, 5-FU or combination drug therapy remains the first-line treatment for gastrointestinal malignancies; however, the overall effective cure rate is insufficient owing to drug resistance [43].Types of 5-FU resistance include primary resistance and secondary resistance.Resistance to chemotherapy in colorectal cancer cells is an important factor affecting clinical treatment failure, tumor recurrence, migration, invasion, and disease deterioration [44,45].It is reported that when 5-FU is combined with other anti-cancer drugs or 5-FU based regimens (FOLFOX, XELOX, and SOX) the response rates of 5-FU based first-line chemotherapy were raised to 40-50% [43].Thus, novel therapeutic strategies are of immediate requirement to combat drug resistance and improve drug response rates.
It has been reported that the combination of traditional Chinese medicine monomer and 5-FU can enhance the sensitivity of chemotherapy.Curcumin, Ginkgo biloba exocarp extracts, and Astragaloside IV reverses 5-FU resistance in various tumors [46][47][48].Exploring new traditional Chinese medicine monomers to enhance chemotherapy sensitivity is expected to provide new ideas for alleviating chemotherapy resistance in clinic.Additionlly, some lncR-NAs are key contributors to various chemoresistance mechanisms and are important determinants of the efficacy of anticancer therapies in cancers.LncRNA HOTAIR contributes to 5-FU resistance through suppressing miR-218 and activating NF-κB/TS signaling in colorectal Cancer [49].LncRNA FGD5-AS1 promotes glycolysis through modulating the miR-330-3p-HK2 axis, leading to 5-Fu resistance of colorectal cancer cells [50].Formononetin, an isoflavonoid isolated from astragalus membranaceus and spatholobus suberectus, relieves the chemotherapy resistance through lncRNA AFAP1-AS1-miR-195/miR-545 axis in triple-negative breast cancer [17].Therefore, the mechanism of Chinese medicine monomer participating in 5-FU resistance by regulating lncRNA is worth studying.Our data suggest that LINC00612 expression is aberrantly upregulated in 5-FU resistant colon carcinoma cells.Silencing LINC00612 expression downregulated the IC50 value of 5-FU resistant colon carcinoma cells, thereby enhancing their chemosensitivity.PSB, a newly discovered traditional Chinese medicine monomer,has attracted attention for its anti-inflammatory, antibacterial and antioxidant properties [20].In recent years, its anti-tumor properties have also been discovered.For instance, PSB can inhibit the proliferation of human bladder cancer cell lines and colon cancer cell lines [19].To be specific, PSB inhibits the proliferation and promotes the apoptosis of human bladder cancer cells via interference with cell cycle regulation [20].PSB inhibits GOLPH3 expression and intracellular signaling pathways in colon cancer cells [19].Whether the inhibitory effect of PSB on colon caner is related to drug resistance is unknown.In this study, we found that PSB suppressed the viability of 5-FU resistant cells and inhibited LINC00612 expression.Hence, we speculate that PSB alleviates drug resistance in colon cancer cells by modulating LNC00612, which was substantiated by our experimental findings.Elevated LINC00612 expression reverses the inhibitory effects of PSB on cell growth.
Meanwhile, lncRNAs can serve as miRNA sponges and act as a new type of competing endogenous RNA (ceRNA) regulator to degrade miRNAs [51,52].Our data demonstrated that LINC00612 could bind to miR-590-3p.miR-590-3p has been shown to affect the biological function of tumor cells in various cancers, including gastric cancer [53], liver cancer [54], and breast cancer [55].However, whether miR-590-3p could affect drug resistance in colon cancer warrants further investigation.GOLPH3, located on chromosome 5p13, is a highly conserved membrane protein in the Golgi complex, which is closely related to structural maintenance, vesicle transport, and Golgi glycosylation [56].GOLPH3 has been identified as an oncoprotein and is significantly upregulated in prostate cancer [57], esophageal cancer [58], gastric cancer [59], ovarian cancer [60], and colon cancer [61].Herein, LINC00612 expression positively regulates GOLPH3 expression.Moreover, GOLPH3 knockdown reversed the effects of LINC00612 on 5-FU-resistance in colon adenocarcinoma cells.These results revealed that PSB modulated LINC00612 to sponge miR-590-3p, thereby suppressing GOLPH3 expression in 5-FU-resistant colon adenocarcinoma cells.
However, the limitations of the present study must be addressed.Herein, we examined colon cancer tissues obtained only from Han Chinese patients; hence, the sample capacity should be broadened, and multiple ethnic groups must be investigated.

Conclusion
The findings of the present study demonstrate the regulation of 5-FU chemoresistance in colon cancer via the LINC00612/ miR-590-3p/GOLPH3 pathway.In addition, we confirmed that PSB attenuated 5-FU chemoresistance in colon cancer by targeting LINC00612, which may provide a novel treatment strategy for 5-FU chemoresistance in colon cancer.

Acknowledgements Not applicable.
Author contributions All authors participated in the design and interpretation of the studies, analysis of the data, and review of the manuscript.Z H and Y L drafted the work and revised it critically for important intellectual content; M C, X C, X D, and Y W were responsible for the acquisition, analysis, or interpretation of data for the work; and C W and C Q made substantial contributions to the conception or design of the work.All authors have read and approved the final manuscript.

Table 1
Primer sequences Primer