Structure–Activity Relationship Study of (E)-3-(6-Fluoro-1H-indol-3-Yl)-2-methyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (FC116) Against Metastatic Colorectal Cancers Resistant to Oxaliplatin

Abstract

Advanced metastatic colorectal cancer (mCRC) and the development of drug resistance to chemotherapy pose significant challenges in clinical settings. In previous studies, we have demonstrated the potent cytotoxic activity of (E)-3-(6-fluoro-1H-indol-3-yl)-2-methyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (FC116) and related 30 derivatives against mCRC by targeting microtubules. In this study, we aimed to evaluate the efficacy of the 31 compounds and explore the structure–activity relationship (SAR) against oxaliplatin-resistant mCRC. We found that most of the derivatives showed high sensitivity toward the oxaliplatin-resistant HCT-116/L cells. Particularly, FC116 exhibited a better GI₅₀ value against the resistant mCRC cell line, HCT-116/L, compared to standard therapies. We also observed a safer therapeutic window for FC116 and a synergistic effect when it was used in combination with oxaliplatin. Mechanistically, FC116 induced the G2/M phase arrest by downregulating cyclin B1 expression through its interaction with microtubules in resistant colorectal cancer cells. Furthermore, in vivo experiments demonstrated that FC116 significantly suppressed tumor growth, achieving a 78% reduction at a dose of 3 mg/kg, which was superior to the 40% reduction achieved by oxaliplatin treatment. Overall, our findings suggest that the indole-chalcone compound FC116 represents a promising lead for chemotherapy in oxaliplatin-resistant mCRC.

Colorectal cancer (CRC) is a prevalent malignant tumor with high mortality rates worldwide.¹⁻³ Despite advancements in early diagnosis and surgical resection, the treatment of CRC in the clinic continues to face significant challenges.^1,2 Over half of the patients are diagnosed in advanced stages, and chemotherapy is the vital therapeutic approach in advanced metastatic CRC (mCRC).⁴⁻⁶ However, drug resistance frequently emerges in mCRC patients, leading to decreased effectiveness of chemotherapy.⁷ Moreover, there are two subtypes of CRC, microsatellite instability (MSI) and microsatellite stability (MSS).^8,9 While MSS CRCs exhibit sensitivity to standard chemotherapy drugs such as 5-fluorouracil, MSI CRCs do not respond well to conventional chemotherapeutic agents. Patients with MSI CRCs often experience a poorer prognosis and shorter overall survival rate.¹⁰⁻¹³ Therefore, there is an urgent need to investigate potential chemotherapeutic agents for MSI CRC patients and those who have developed resistance to standard therapies.

Chalcone (Figure 1), a widely existing α,β-unsaturated ketone natural product, has confirmed to possess a broad spectrum of biological activities.¹⁴ Applying this classical chemistry template, a series of photoaffinity labeling clickable probes (e.g., C95, Figure 1) were developed by our group.¹⁵ The anticancer target identification was performed to confirm the target protein as β-tubulin, located near the colchicine-binding site. Further structure–relationship relationship (SAR) study discovered the α-methyl group and trimethoxylphenyl group as the anticancer pharmacophores.^16,17 The indole moiety was introduced to replace the traditional phenyl group to significantly improve the anticancer potency to a signal-digit nanomolar range. For instance, FC77 (Figure 1), an indole-chalcone derivative, was confirmed to possess high potency against an NCI-60 cancer cell line panel (average GI₅₀ < 6 nM).¹⁷ Toward the multidrug-resistant CRC cell line (HCT116/L, resistant to oxaliplatin), FC77 still showed high sensitivity (GI₅₀ = ∼ 6 nM). However, low selectivity toward normal cells leading to high toxicity in tumor models was demonstrated. Further SAR study generated the discovery of a representative fluoro-substituted indole-chalcone (FC116, Figure 1).^16,18 This compound has demonstrated high potency against HCT116 and CT26 cell lines, with IC₅₀ values of 4.52 and 18.69 nM, respectively. In HCT116-xenograft mice, FC116 exhibited a tumor growth inhibition rate of 65.96% at a dose of 3 mg/kg, which is much better than paclitaxel (PTX). In APC^min/+ mice, FC116 showed a significant adenoma number inhibition rate of 76.3%.¹⁶ Additional modifications on the 4-methoxyphenyl group led to a water-soluble compound FC11619 (Figure 1) that achieved tumor growth inhibition rates of 65.3% and 73.4% at doses of 5 and 10 mg/kg/d for 21 d.¹⁸

Figure 1.

Chemical structures of chalcones and the representative chalcones developed by our group.

Therefore, indole-chalcone derivatives should be a kind of privileged compound for anticancer study. However, the efficacy of the series of indole-chalcones against resistance to standard therapies has not been investigated. Oxaliplatin-based chemotherapy is the current standard for mCRC, and acquired resistance to oxaliplatin eventually occurs and is still a major cause of treatment failure.^19,20 In this study, we applied 31 indole-based chalcones previously published by our group¹⁶ to further determine their efficacy and explore the SAR against oxaliplatin-resistant mCRC. We found that most of the derivatives showed high sensitivity toward the oxaliplatin-resistant HCT-116/L cells, particularly, FC116. Comparison to standard therapies and tumor growth inhibition using the colorectal cancer xenograft mice model as well as the molecular mechanisms were investigated.

2. Results and Discussion

2.1. The Indole-Chalcone Analogs Displayed Great Cytotoxicity Against the Resistant HCT-116 Cell/L and a Structure–Activity Relationship (SAR) Was Concluded

The synthetic aspects and the activity against unresistant mCRC cell line HCT-116 have been determined in our previous study.¹⁶ The indole moiety of FC77 was unchanged, and the SAR against the resistant cell line HCT-116/L (resistant to oxaliplatin) on the left phenyl group was determined (Table 1). Oxaliplatin was used as a control drug, showing a GI₅₀ of 5314 nM against HCT-116 and resistant to HCT-116/L with a GI₅₀ of 77 213 μM. Compound 1 displayed a GI₅₀ much lower than that of FC77 in ∼1000-fold, highlighting the importance of 3,4,5-trimenthoxy substitution in the indole-chalcone template. The α-methyl was critical to the activity, as determined by compound 2. Compound 3 with nonsubstitution on 4-position showed a GI₅₀ of 14 nM against HCT-116/L. Compound 4 with 3,4-dimethoxy group decreased the cytotoxicity to 84 nM, and compound 5 with 3-methoxy group showed a GI₅₀ of only 194 nM. Compounds 3–6 were generated to confirm the influence of the trimethyoxy, indicating the activity on the sequence: 3OCH₃ > 2OCH₃ > OCH₃. Compound 6 had a 1,3-dioxolane that kept the activity with a GI₅₀ of 17 nM. They together indicated the 3-, 5-OCH₃ as the key pharmacophores. Compound 7 contains a pyridinyl that had a low cytotoxicity against the cells. Compound 8 with an α-ethyl showed about 2-fold decrease in GI₅₀ as 13 nM against HCT-116/L. Compound 9 with a 3,4-dihydronaphthalenone showed no activity at 10 000 nM.

Table 1. In Vitro Cytotoxicity of Analogs (1–9) Against the HCT-116/L Cell Line^a.

^aGI₅₀, concentration causing 50% inhibition of the cell growth determined by CellTiter Blue assay. All experiments were repeated independently at least three times. The GI₅₀ values are presented as the means ± standard error (S.E.M.).

Next, the substitutions on the indole were further investigated (Table 2). First, a methyl was tried on the indole (compounds 10–13). The methyl on 4-position obtained compound 10 that showed a low cytotoxicity against HCT-116/L (GI₅₀ = 141 nM). Compounds 11 and 12 with a 5- and 6-methyl displayed similar GI₅₀s (GI₅₀ =30, 24 nM). Introducing a methyl on 7-position obtained compound 13 that showed the best activity (GI₅₀ = 16 nM) in this series. Next, the halogen compounds were synthesized. Compounds 14 and 15 (named FC116 in our group) with 5-, 6-fluoro group exhibited high activity with GI₅₀ of 7 nM and 6 nM. Compound 16 with a 5-chloro was very potent (HCT-116/L, GI₅₀ = 17 nM). However, compound 17 with a relatively large bromo lost the activity at 10 μM (GI₅₀ > 10 μM). Third, a strong electron-withdrawing group (NO₂) was tried, indicating decreased activity compared to FC77. Compound 18 showed a 10-fold decrease with a GI₅₀ of 71 nM. Compound 19 with 6-NO₂ and compound 20 with a 7-NO₂ still showed the high potency against the resistant HCT-116/L cell (GI₅₀ = 15 and 24 nM). When the nitro group was reduced to an amino group, compound 21 decreased the cytotoxicity in one-fold. Finally, encouraged by FC116 with a 6-substitution, we tried methyloxyl (22), ester (23), and carboxyl (24) on the position. Compound 22 had GI₅₀ values of 47 nM against HCT-116/L. Compound 23 made the activity decreased to 151 nM. Compound 24 totally lost the cytotoxicity at 10 μM.

Table 2. In Vitro Cytotoxicity of Analogs (10–24) Against the HCT-116/L Cell Line.

compounds	R3	R4	R5	R6	GI50(nM)
10	CH3	H	H	H	141 ± 27
11	H	CH3	H	H	30 ± 5
12	H	H	CH3	H	24 ± 5
13	H	H	H	CH3	16 ± 3
14	H	F	H	H	7 ± 1
15 (FC116)	H	H	F	H	6 ± 1
16	H	Cl	H	H	17 ± 2
17	H	Br	H	H	>10000
18	H	NO2	H	H	71 ± 5
19	H	H	NO2	H	15 ± 1
20	H	H	H	NO2	24 ± 3
21	H	H	NH2	H	27 ± 3
22	H	H	OCH3	H	47 ± 2
23	H	H	COOCH3	H	151 ± 19
24	H	H	COOH	H	>10000
oxaliplatin					77213 ± 2041

Last, the whole indole part was replaced by using different heterocycles (Table 3). Compounds 25–30 were obtained using pyridine to change the benzyl ring of the indole. The small nitrogen made the varying activity, indicating the importance of the nitrogen in the template. Compound 25 with 2-N-pyridine and 28 with 5-N-pyridine showed better potency in the low nanomolar range. Compound 26 with 3-N-pyridine and 27 with 4-N-pyridine dramatically decreased the potency to GI₅₀ values of 386 and 595 nM. Compound 29 with an imidazo[1,2-a]pyridine group had a low GI₅₀ value of 44 nM against HCT-116/L. Compound 31 with a phenylthiophenol was still active with a GI₅₀ value of 27 nM against HCT-116/L.

Table 3. In Vitro Cytotoxicity of Analogs (25–31) Against the HCT-116/L Cell Line.

To sum up, the indole chalcones showed sensitivity to the resistant cell line like nonresistant cell HCT-116.¹⁶ This study again highlighted the importance of the trimethoxy and α-methyl substitutions. The 6-substitutions on the indole-benzyl ring favored the cytotoxicity. The pyridine and thiophene might be useful to replace the benzyl ring; however, the position of the nitrogen in the pyridine should be taken into consideration.

2.2. FC116 Showed Better Sensitivity Toward the Resistant mCRC Cell Line HCT-116/L Compared with the Standard Therapies

Fluorine-containing compounds are recognized to be a substantial proportion of therapeutically useful drugs and drug candidates.^21,22 Among the analogs, FC116 with a fluoro group exhibited the best activity with a GI₅₀ of 6 nM against HCT-116/L, which was about 14-fold more sensitive toward the nonresistant HCT-116 cells (resistance fold = 0.076). Considering that the cytotoxic chemotherapy is the standard therapy for mCRC, we evaluated the sensitivity of HCT-116 and HCT-116/L cells to a variety of standard therapies including oxaliplatin, doxorubicin, cytarabine, mitoxantrone, vinblastine, and vorinostat, as well as FC77 and FC116 for comparison (Table 4). As a result, compared with unresistant cells, only vorinostat and FC116 were demonstrated to be sensitive toward the resistant cell HCT-116/L. The other compounds showed cross-resistance to cells in different trends. Even the compounds vinblastine (12-fold) and FC77 (6-fold) targeting microtubule were still resistant to the oxaliplatin-resistant CRC cell line. Although cytarabine was sensitive toward HCT-116/L than HCT-116, the potency was only at a submicromolar level, which was much lower than the FC116. These data further indicated the potential of FC116 as a unique candidate to treat mCRC, especially the related drug-resistant cancers. However, as a cytotoxic compound, the side effects cannot be ignored as the lead compound FC77 has a low selectivity toward normal cells, leading to high toxicity.¹⁷

Table 4. Comparison of the Standard Therapies FC77 and FC116^a.

	GI50(nM)
compounds	HCT116/L	HCT116	resistance fold
oxaliplatin	77213 ± 2041	5314 ± 346	14.53
doxorubicin	450 ± 220	440 ± 50	1.02
cytarabine	3380 ± 1400	84390 ± 15990	0.04
mitoxantrone	1460 ± 110	1030 ± 210	1.42
vinblastine	60 ± 20	5 ± 3	12
vorinostat	2130 ± 760	2230 ± 370	0.96
FC77	6 ± 3	1 ± 0	6
FC116	6 ± 1	79 ±7	0.076

^aThe ratio of GI₅₀, HCT-116/L / GI₅₀, HCT-116.

2.3. FC116 Showed a Low Toxic Side Effect Toward Normal Human Intestinal Epithelial Cells and a Safer Therapeutic Window

The cytotoxicity and therapeutic window of FC116 against HCT-116, HCT-116/L, and normal human intestinal epithelial (HIEC) cells were further evaluated (Figure 2). The viability of HCT-116 and HCT-116/L cells was significantly and dose dependently reduced upon treatment with this compound. FC116 was more active against drug-resistant HCT-116/L cells than against HCT-116 cells in the three dosages (0.01, 0.1, and 1 μM). As expected, the conventional chemotherapeutic agent oxaliplatin for CRC was active against HCT-116 cells but less effective against the resistant HCT-116/L cell line. Doxorubicin was sensitive to the two mCRC cell lines. While oxaliplatin and doxorubicin showed a greater inhibitory effect and no significant difference by normal HIEC than by mCRC cells, FC116 was markedly less toxic to normal HIEC than to mCRC cells at the same treating concentrations, suggesting its safer therapeutic window and translational potential.

Figure 2.

Preferential cytotoxicity of FC116 to HCT-116 and HCT-116/L and its therapeutic window for HIEC. *p < 0.05, **p < 0.01.

2.4. FC116 Exhibited Synergistic Effect with CRC Standard Therapy Oxaliplatin

We tested the potential synergism of FC116 in combination with oxaliplatin in mCRC cell lines (Figure 3). The combination index (CI) was calculated using the Chou and Talalay method.²³ Our results showed that the combination of oxaliplatin and FC116 enhanced the inhibition compared with individual compounds. For the resistant HCT116/L cells (Figure 3A), the CI values for the combination of oxaliplatin with FC116 at 5 nM were 0.3–0.47, suggesting strongly synergistic interaction. The CI values were 0.48 and 0.68 at concentrations of 1 nM FC116 (11.1 and 33.3 μM for oxaliplatin), suggesting synergism; and the CI value was 0.29 at 100 μM for oxaliplatin, suggesting strong synergism. For HCT116 cells (Figure 3B), the combination shows synergism only at lower concentrations (0.6 μM and 1.8 μM for oxaliplatin; 5 nM for FC116) with the CI of 0.37 and 0.60. The results suggested that FC116 can be used in combination with standard therapies to treat mCRC cells.

Figure 3.

Inhibitory effect of the combination of oxaliplatin and FC116 (1 nM, 5 nM) against HCT116/L and HCT116 cells. CI values above data points were calculated by CompuSyn software. CI < 0.9, CI = 0.9–1.1, and CI > 1.1 indicate synergism, additive effect, and antagonism, respectively. Data are expressed as mean ± standard error (S.E.M.).

2.5. FC116 Inhibit Tubulin Polymerization In Vitro

Based on our previous result, the chalcone compound and FC116 indicated a colchicine-like microtubule-depolymerizing mechanism.¹⁶⁻¹⁸ Moreover, it is reported that tubulin-binding agents that specifically target the colchicine binding site may circumvent the multidrug resistance.²⁴ A chalcone probe has been previously determined to target N337–K350 residues nearby the colchicine-binding site by a mass spectrometry-based approach.¹⁵ Therefore, as a chalcone analog, the effect of FC116 on the microtubule networks was confirmed using an immunofluorescent assay. As shown in Figure 4, HCT116 and HCT116/L cells in the control group exhibited normal organization. However, after treatment with FC116 (4 and 8 nM) for 24 h, the microtubule networks were decreased and disorganized, which indicated that the compound FC116 could strongly inhibit tubulin polymerization at low concentrations.

Figure 4.

Immunofluorescence assay on the microtubule network of HCT116/L and HCT116 treated with FC116. HCT116/L and HCT116 cells were treated with control 0.1% DMSO, FC116 (4 and 8 nM). Then, the cells were fixed and stained with anti-α-tubulin-FITC antibody (green) and Alexa Fluor 488 dye and counterstained with DAPI (blue). The detection of the fixed and stained cells was performed with a TCS SP5 II laser confocal microscope (Leica, Germany).

2.6. FC116-Induced Cell Cycle Arrest in G2/M Phase

G2/M phase cell cycle arrest is a key feature of antimicrotubule agent-introduced cytotoxicity.¹⁶⁻¹⁸ We therefore characterized the impact of FC116 on the cell cycle distribution using the two mCRC cell lines (Figure 5). Flow cytometry showed that cells treated with FC116 significantly increased population in G2/M phases relative to the control in a dose–response manner, consistent with their antimicrotubule mechanism of action. Furthermore, cyclin B1, a marker protein for G2/M,²⁵ was examined (Figure 5C,D). Compared with the flow cytometry tests, the Western blotting assay is less sensitive to the detection of cell cycle-related proteins. Therefore, higher concentrations of FC116 might be required for cyclin B1 detection. First, we tried different higher concentrations (0.02, 0.2, and 2 μM) of FC116 at 6 h in HCT116/L cells. As a result, Figure 5C did not show apparent difference for the protein. Then, we used the highest concentration (2 μM) at different time points (12, 24, and 48 h) in HCT116/L cells. Western blotting assay showed that the expression level of cyclin B1 was subsequently downregulated in a time-dependent manner and nearly disappeared after 48 h in HCT116/L cells (Figure 5D).

Figure 5.

Effect of FC116 on cell cycle distribution toward (A) HCT116/L and (B) HCT116 cells at different concentrations (HCT116/L: 0, 3, 6, and 12 nM; HCT116: 0, 2, 5, and 10 nM) for 24 h. *p < 0.05, **p < 0.01 versus G2/M phase of the control group. All the experiments are triplicated. (C) Effect of FC116 on the expression level of the G2/M phase marker protein cyclin B1 by Western blotting at different concentrations (0.02, 0.2, and 2 μM) at 6 h in HCT116/L cells. (D) Effect of FC116 on the expression level of the G2/M phase marker protein cyclin B1 by Western blotting at different time points (12, 24, and 48 h) in HCT116/L cells.

2.7. FC116 Inhibited Colorectal Tumor Growth In Vivo

To investigate the antitumor potency of FC116 in vivo, a colorectal cancer xenograft mice model was established (Figure 6). The mice were administered 1.5 mg/kg/day and 3 mg/kg/day of FC116, and 10 mg/kg/day of oxaliplatin (positive control) and the vehicle (negative control) by intraperitoneal injection as previous studies. As shown in Figure 6A, FC116 displayed obvious and dose-dependent antitumor activity. The decrease in tumor weight reached 78% at the dose of 3 mg/kg/day of FC116 (Figure 6B,D) when compared to vehicle at 21 days after the initiation of treatment. By contrast, oxaliplatin (inhibition rate: 40% at much higher dosage, 10 mg/kg/day) is less potent than FC116 (3 mg/kg) and comparable to FC116 (45%, at a dose of 1.5 mg/kg). This result further confirmed the in vitro result on the resistance of oxaliplatin. Furthermore, FC116 did not cause an obvious loss of body weight in these mice treated with FC116 (Figure 6C), which demonstrated that FC116 displayed no significant toxicity. All of these results confirmed that FC116 exhibited superior antitumor activity in vivo, especially in oxaliplatin-resistant cancers.

Figure 6.

FC116 inhibited colorectal tumor growth in vivo. Nude mice bearing HCT116/L cells were administered with oxaliplatin (10 mg/kg/day), FC116 (1.5, 3 mg/kg/day) and vehicle for 3 weeks, the mice were sacrificed, and the tumors were weighted. Figures present the changes of (A) tumor volume, (B) tumor weight, (C) body weight, (D) tumors of each group from mice at 21 days after initiation of treatment. *p < 0.05, ***p < 0.001 vs control group.

3. Conclusion

In this study, 31 synthesized indole-chalcone-based compounds were applied to evaluate their efficacy against advanced mCRC drug resistance. The SAR was summarized for this unique chemical template. They overall displayed superior potent cytotoxicity and sensitivity toward resistant colorectal cancer cells. They still acted as microtubule depolymerization agents and significantly induced cancer cells arrested in G2/M phase arrest via downregulating cyclin B1 expression. In vivo, FC116 significantly suppressed tumor growth, achieving 78% at the dose of 3 mg/kg, which was much better than standard therapy oxaliplatin. Collectively, the indole-chalcones represented a lead compound for discovering chemotherapeutic of resistant metastatic colorectal cancers. However, we still do not know the long-term therapeutic viability of this kind of colchicine-like microtubule-depolymerizing agent for overcoming resistance. Applying the highly potent compounds in proteolysis-targeting chimeras (PROTACs) might be a future direction.

4. Experimental Section

4.1. Chemistry

All the compounds were obtained using the same procedures in our previous publication.¹⁶ All final compounds exhibited purities of >95% for biological experiments.

4.2. Cell Culture

HCT116 and HIEC cells were purchased from ATCC. All of cell lines were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in an atmosphere of 5% CO₂ at 37 °C. Initially, oxaliplatin (100 ng/mL) was used to induce drug resistance in HCT116 cells, and the concentration of oxaliplatin was gradually increased for about 8 months. Then, the cells, named the HCT116/L cell line, could grow stably. Oxaliplatin (10 μg/mL) was added to the medium of HCT116/L cells to maintain resistance.^26,27

4.3. In Vitro Cytotoxicity

The cells were plated in a 96-well plate (about 5000 cells/well) and treated with a series of dilutions of the test compounds with a 72 h treatment. The evaluation was achieved using a CellTiter-Blue cell viability assay kit, and the GI₅₀ values of each compound were determined by fitting the relative cell viability to the drug concentrations using GraphPad Prism 6.0 (GraphPad software, San Diego, CA, USA). The reported GI₅₀ values are the average of at least three independent experiments.

4.4. Analysis of Cell Cycle Distribution by Flow Cytometry

HCT116 and HCT116/L cells were plated in six-well plates (1 × 10⁶ per well). After 24 h, the cells were treated with various concentrations of FC116 (HCT116/L: 0, 3, 6, 12 nM; HCT116: 0, 5, 10, 20 nM) in RPMI complete medium. After 24 h treatment, the cells were harvested by centrifugation at 1000 rpm for 5 min. The cell pellet was resuspended in 70% (v/v) ethanol and stored at 4 °C overnight. Cell pellets were collected by centrifugation, washed twice with PBS, and then suspended in 500 mL phosphate-buffered saline (PBS) containing 0.02 mg/mL propidium iodide (PI) and 0.1 mg/mL ribonuclease A (RNase A) in darkness at 37 °C for 30 min. The cell cycle distribution was then analyzed using a BD FACSCalibur flow cytometer.

4.5. Western Blot Analysis

HCT116/L cells were seeded in six-well plates (1 × 10⁶ per well) and grown until 70–80% confluence. Cells were treated with FC116 for 12 h, 24 h, and 48 h at 37 °C. After treatment, cells were harvested and centrifuged at 1300 rpm for 5 min. Then cells were washed twice with PBS and lysed on ice for 30 min with RIPA lysis buffer containing protease and phosphatase inhibitors. After centrifugation at 14 500 rpm for 20 min, the supernatants were transferred to new tubes and stored at −80 °C. A BCA protein assay kit (Thermo Fisher Scientific) was used to quantify the proteins. Equal amounts of protein were resolved by SDS/PAGE and electrotransferred onto PVDF membranes. The membranes were blocked with 5% skim milk in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) for 1 h and then incubated with primary antibodies to cyclin B1 (Abcam, ab32053, 1:3000), GAPDH (Abcam, ab9485, 1:4000) at 4 °C overnight. Subsequently, the membranes were washed with TBST for 15 min and then probed with appropriate secondary antibodies conjugated with horseradish peroxidase for 2 h, washed three times with TBST, and bands were visualized by a LI-COR gel imaging system (Odyssey, USA).

4.6. Immunofluorescence Assay

After being cultured in 6-well plates (2.5 × 10⁵ per well), HCT116 and HCT116/L cells were treated with 4, 8 nM FC116 or 1% DMSO. After 24 h, cells were fixed with 4% paraformaldehyde for 10 min and washed with ice PBS for 3 times. Subsequently, the samples were incubated with PBS (containing 0.25% Triton X-100) for 10 min. Then, the cells were blocked with QuickBlockTM immunostaining sealant (biyuntian, P0260,1:500) for 30 min and incubated with primary antibody (Abcam, ab179484,1:250) at 4 °C overnight. Cells were incubated with secondary antibodies (biyuntian, Alexa Fluor 488,1:500) for 1 h at room temperature and restained with DAPI (biyuntian, C1005) for 1 min. Finally, the cells were imaged with fluorescence confocal microscopy (Leica TCS SP5II, germany).

4.7. In Vivo Antitumor Activity Assay

Female nude mice (5–6 weeks old) were injected with 5 × 10⁶ cells/200 μL of HCT116/L cells subcutaneously into the right hind flank. After 10 days of transplantation, the mice were randomly assigned to four groups. The test compounds were suspended in normal saline containing 0.5% carboxymethyl cellulose sodium, 3% Tween-80, and 1% castor oil. Different formulations were injected in the intraperitoneal of these mice according to the following groups: Group 1, OXA (10 mg/kg/d); Group 2, FC116 (3 mg/kg/d); Group 3, FC116 (1.5 mg/kg/d); and Group 4, solvent (control). At the end of 21 days, mice were sacrificed. Tumor volumes were calculated using the equation: volume (cm³) = width² (cm²) × length (cm)/2. The animal experiment was performed following the guidance of the Animal Care and Use Committee of Second Military Medical University.

4.8. Statistical Analysis

Each experiment was performed at least three times and analyzed. The data are expressed as means ± standard error (S.E.M.). Comparisons were made using a two-tailed Student t test or analysis of variance (ANOVA). A value of p < 0.05 was considered statistically significant.

Glossary

Abbreviations

CI

combination index

CRC

colorectal cancer

FBS

fetal bovine serum

HIEC

normal human intestinal epithelial

mCRC

metastatic colorectal cancer

MSI

microsatellite instability

MSS

microsatellite stability

PBS

phosphate-buffered saline

PI

propidium iodide

PROTACs

proteolysis-targeting chimeras

RNase A

Ribonuclease A

SAR

structure–activity relationship

Author Contributions

^# S.W. and Q.G. These authors contributed equally.

The authors declare no competing financial interest.