Cytotoxic Compounds from Belamcanda chinensis (L.) DC Induced Apoptosis in Triple-Negative Breast Cancer Cells

Four compounds (1, 5, 7, and 8) were first isolated from the genus Belamcanda Adans. nom. conserv., and six known compounds (2–4, 6, 9, and 10) were isolated from the rhizome of Belamcanda chinensis (L.) DC. Their structures were confirmed by spectroscopic data. Herein, compounds 1–10 were rhapontigenin, trans-resveratrol, 5,7,4′-trihydroxy-6,3′,5′-trimethoxy-isoflavone, irisflorentin, 6-hydroxybiochannin A, iridin S, pinoresinol, 31-norsysloartanol, isoiridogermanal, and iristectorene B, respectively. All compounds were evaluated for their antiproliferative effects against five tumor cell lines (BT549, 4T1, MCF7, MDA-MB-231, and MDA-MB-468). Among them, compound 9 (an iridal-type triterpenoid) showed the highest activity against 4T1 and MDA-MB-468 cells. Further studies displayed that compound 9 inhibited cell metastasis, induced cells cycle arrest in the G1 phase, exhibited significant mitochondrial damage in 4T1 and MDA-MB-468 cells including excess reactive oxygen species, decreased mitochondrial membrane potential, and induced 4T1 and MDA-MB-468 cell apoptosis for the first time. In summary, these findings demonstrate that compound 9 exerts promising potential for triple-negative breast cancer treatment and deserves further evaluation.


Introduction
Belamcanda chinensis (L.) DC is the sole species in the genus Belamcanda Adans. nom. conserv., native to Southeast Asia (Bhutan, China, Japan, Korea, Myanmar, the Philippines, Thailand, the Ussuri region of Russia, and Vietnam), and it is widely distributed in Hubei, Henan, Jiangsu, and Anhui in China [1]. The rhizome of Belamcanda chinensis (L.) DC was introduced as a medicine in the first Chinese monograph on herbal medicines, Shennong's Classic of Materia Medica [2]. It is known as "She Gan" in China, and its characteristics are that it is bitter and cold, related to the lungs, with sinking and dispersing action, and descending lung "qi". It treats a sore throat by eliminating heat, toxins and phlegm, dyspnea, dissolves sputum, and relieves wheezing caused by the accumulation of heat and phlegm in the lungs [3][4][5][6][7].

Effects of Compounds 1-10 on the Proliferation of Breast Cancer Cells
The isolated compounds were evaluated for their antiproliferation effects on BT549, 4T1, MDA-MB-468, MDA-MB-231, and MCF7 cell lines treated with 50 µM by CCK-8 assay ( Figure 2). The results show the different antiproliferation effects of compounds 1-10 on five kinds of breast cancer cells. Notably, compounds 9 and 10 were determined for the cytotoxicity of breast cancer cells for the first time. Among five kinds of breast cancer cells, compound 9 displayed the highest activity against the BT549, 4T1 MDA-MB-468, and MCF7 cell lines. In addition, the cytotoxicity of compound 10 clearly became weaker than compound 9 as a result of the substituent group of the long fatty chain. Consequently, compound 9 was chosen for subsequent mechanistic studies.
To verify the above results, we conducted a colony formation assay to prove the effects of compound 9 on the proliferation of the MDA-MB-468 and 4T1 cells. The colony formation assay revealed treatment with compound 9 of 30 µM in the MDA-MB-468 and 4T1 cells compared with the control group could significantly reduce the overall number and size of colonies formed (p < 0.001) ( Figure 3C). To further confirm the inhibitory effect of compound 9 on cell proliferation, we performed a cell cycle assay. Cells prepare for DNA replication in the G1 phase; the G2/M phase continuously allows preparation for mitosis. During the process of development, differentiation, or regression of growth factors, cells can enter the inactive phase G0 and then recover to G1 [60]. We detected the cell cycle by propidium iodide staining (PI staining). After the DNA inside the cells was stained with PI, the DNA content of the cells could be measured using flow cytometry. Then, based on the distribution of DNA content, cell cycle analysis can be performed. Compared with the control group, treatment with compound 9 of 20 or 30 µM in the MDA-MB-468 and 4T1 cells could prominently accumulate the cells in the G1 phase and decrease the S phase ( Figure 3D). In summary, these results demonstrated that compound 9 inhibited the growth of MDA-MB-468 and 4T1 cells.
To verify the above results, we conducted a colony formation assay to prove the effects of compound 9 on the proliferation of the MDA-MB-468 and 4T1 cells. The colony formation assay revealed treatment with compound 9 of 30 µM in the MDA-MB-468 and 4T1 cells compared with the control group could significantly reduce the overall number and size of colonies formed (p < 0.001) ( Figure 3C). To further confirm the inhibitory effect of compound 9 on cell proliferation, we performed a cell cycle assay. Cells prepare for DNA replication in the G1 phase; the G2/M phase continuously allows preparation for mitosis. During the process of development, differentiation, or regression of growth factors, cells can enter the inactive phase G0 and then recover to G1 [60]. We detected the cell cycle by propidium iodide staining (PI staining). After the DNA inside the cells was stained with PI, the DNA content of the cells could be measured using flow cytometry. Then, based on the distribution of DNA content, cell cycle analysis can be performed. Compared with the control group, treatment with compound 9 of 20 or 30 µM in the MDA-MB-468 and 4T1 cells could prominently accumulate the cells in the G1 phase and decrease the S phase ( Figure 3D). In summary, these results demonstrated that compound 9 inhibited the growth of MDA-MB-468 and 4T1 cells.

Compound 9 Suppressed Metastasis in the TNBC Cells
Tumor metastasis is the main cause of poor prognosis in TNBC patients and poses a serious threat to them. Consequently, we conducted wound-healing assay and transwell assays to assess whether compound 9 was able to suppress MDA-MB-468 and 4T1 cell metastasis. As shown in the wound-healing assay, treatment with compound 9 of 30 µM in the MDA-MB-468 cells compared with the control group could significantly increase the percentage wound closure (%) (p < 0.001), but treatment with compound 9 of 20 µM in the 4T1 cells could markedly increase the percentage wound closure (%) (p < 0.001). In addition, the higher the concentration or the longer the treatment time of compound 9, the higher the percentage of wound closure (%), which revealed that compound 9 could dose-and time-dependently inhibit migration in the MDA-MB-468 and 4T1 cells ( Figure  4A). Similarly, the results of the transwell assay confirmed that compound 9 of 10 µM could significantly decrease the number of invaded MDA-MB-468 and 4T1 cells (p < 0.001) ( Figure 4B), and compound 9 could dose-dependently inhibit invasion. In brief, these

Compound 9 Suppressed Metastasis in the TNBC Cells
Tumor metastasis is the main cause of poor prognosis in TNBC patients and poses a serious threat to them. Consequently, we conducted wound-healing assay and transwell assays to assess whether compound 9 was able to suppress MDA-MB-468 and 4T1 cell metastasis. As shown in the wound-healing assay, treatment with compound 9 of 30 µM in the MDA-MB-468 cells compared with the control group could significantly increase the percentage wound closure (%) (p < 0.001), but treatment with compound 9 of 20 µM in the 4T1 cells could markedly increase the percentage wound closure (%) (p < 0.001). In addition, the higher the concentration or the longer the treatment time of compound 9, the higher the percentage of wound closure (%), which revealed that compound 9 could dose-and timedependently inhibit migration in the MDA-MB-468 and 4T1 cells ( Figure 4A). Similarly, the results of the transwell assay confirmed that compound 9 of 10 µM could significantly decrease the number of invaded MDA-MB-468 and 4T1 cells (p < 0.001) ( Figure 4B), and compound 9 could dose-dependently inhibit invasion. In brief, these results demonstrated that compound 9 suppressed metastasis in the MDA-MB-468 and 4T1 cells.

Effect of Compound 9 on TNBC Cell Apoptosis
Apoptosis, programmed cell death, regulates the balance between cell growth and death. Tumor cells that evade apoptosis could promote metastasis [61]. Accordingly, we investigated cell apoptosis detection via Annexin V-FITC/PI staining and Hoechst staining. The Annexin V-FITC/PI staining detection kit makes use of FITC-labeled Annexin V as a probe to detect the occurrence of early cell apoptosis. PI cannot penetrate the intact cell membrane of normal or early apoptotic cells but can pass through the cell membrane of late apoptotic and necrotic cells. Therefore, PI is used to distinguish between surviving early cells and necrotic or late apoptotic cells. Hoechst 33342 staining is a solution suitable for staining the nucleus of living cells. The very bright blue fluorescence staining of the nucleus can be observed by a fluorescence microscope.

Effect of Compound 9 on TNBC Cell Apoptosis
Apoptosis, programmed cell death, regulates the balance between cell growth and death. Tumor cells that evade apoptosis could promote metastasis [61]. Accordingly, we investigated cell apoptosis detection via Annexin V-FITC/PI staining and Hoechst staining. The Annexin V-FITC/PI staining detection kit makes use of FITC-labeled Annexin V as a probe to detect the occurrence of early cell apoptosis. PI cannot penetrate the intact cell membrane of normal or early apoptotic cells but can pass through the cell membrane of late apoptotic and necrotic cells. Therefore, PI is used to distinguish between surviving early cells and necrotic or late apoptotic cells. Hoechst 33342 staining is a solution suitable for staining the nucleus of living cells. The very bright blue fluorescence staining of the nucleus can be observed by a fluorescence microscope.
In comparison with the control group, after treatment with compound 9 of 10 µM to 30 µM, the percentage of MDA-MB-468 and 4T1 cells in PI-and Annexin-V-FITC-positive cells was significantly increased via flow cytometry, which demonstrated that the apoptotic rate of the MDA-MB-468 and 4T1 cells was significantly increased (p < 0.001) ( Figure 5A). The results indicated that compound 9 dose-dependently induced both early and late apoptosis in the MDA-MB-468 and 4T1 cells. Moreover, the results of Hoechst 33342 staining observed by a fluorescence microscope were consistent with Annexin V-FITC/PI staining. Compared with the control group, after treatment with compound 9 of 10 µM to 30 µM, the bright blue fluorescence became stronger and underwent typical morphological changes such as nucleus condensing and fragmentation ( Figure 5B) in the MDA-MB-468 and 4T1 cells, which revealed that compound 9 could induce cell apoptosis. Overall, these results showed that compound 9 dose-dependently induced MDA-MB-468 and 4T1 cell apoptosis. In comparison with the control group, after treatment with compound 9 of 10 µM to 30 µM, the percentage of MDA-MB-468 and 4T1 cells in PI-and Annexin-V-FITC-positive cells was significantly increased via flow cytometry, which demonstrated that the apoptotic rate of the MDA-MB-468 and 4T1 cells was significantly increased (p < 0.001) ( Figure 5A). The results indicated that compound 9 dose-dependently induced both early and late apoptosis in the MDA-MB-468 and 4T1 cells. Moreover, the results of Hoechst 33342 staining observed by a fluorescence microscope were consistent with Annexin V-FITC/PI staining. Compared with the control group, after treatment with compound 9 of 10 µM to 30 µM, the bright blue fluorescence became stronger and underwent typical morphological changes such as nucleus condensing and fragmentation ( Figure 5B) in the MDA-MB-468 and 4T1 cells, which revealed that compound 9 could induce cell apoptosis. Overall, these results showed that compound 9 dose-dependently induced MDA-MB-468 and 4T1 cell apoptosis.   from red fluorescence to green fluorescence, and the change of JC-1 from red fluorescence to green fluorescence could also be used as a characteristic sign of cells' early apoptosis.
Compared to the control group, the JC-1 staining assay demonstrated that after treatment with compound 9 of 10 µM to 30 µM in the MDA-MB-468 and 4T1 cells, the red fluorescence became weaker, whereas, on the contrary, the green fluorescence became stronger (Figure 6), which revealed that the MMP levels decreased. As a consequence, these results demonstrated that compound 9 could dose-dependently disrupt MMP, which suggested that compound 9 induced early apoptosis in the MDA-MB-468 and 4T1 cells.

Effect of Compound 9 on ROS Level
Reactive oxygen species (ROS) are a heterogeneous group of highly reactive ions and molecules that are produced by metabolic reactions in mitochondria. ROS maintain homeostasis through the redox pathway, and increasing ROS levels are able to promote tumor cell death and limit cancer progression [62]. We investigated ROS level via an ROS assay kit, which is a reagent kit that makes use of a fluorescent probe of 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) for ROS detection. DCFH-DA itself does not have fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by esterases inside the cell to generate DCFH. However, DCFH cannot penetrate the cell membrane, making it easy for the probe to be loaded into

Effect of Compound 9 on ROS Level
Reactive oxygen species (ROS) are a heterogeneous group of highly reactive ions and molecules that are produced by metabolic reactions in mitochondria. ROS maintain homeostasis through the redox pathway, and increasing ROS levels are able to promote tumor cell death and limit cancer progression [62]. We investigated ROS level via an ROS assay kit, which is a reagent kit that makes use of a fluorescent probe of 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) for ROS detection. DCFH-DA itself does not have fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by esterases inside the cell to generate DCFH. However, DCFH cannot penetrate the cell membrane, making it easy for the probe to be loaded into the cell. Reactive oxygen species within cells can oxidize DCFH to produce fluorescent DCF. By detecting the fluorescence of DCF, the level of intracellular ROS can be determined.
DCF fluorescence demonstrated that at higher concentrations of treatment of compound 9 in the MDA-MB-468 and 4T1 cells through flow cytometry, the fluorescence became stronger which indicated that the ROS levels were higher ( Figure 7A). In detail, compound 9 of 30 µM in the MDA-MB-468 cells and 20 µM in the 4T1 cells significantly increased the ROS levels (p < 0.001) compared to the control group. In addition, the results from the fluorescence microscope were identical; DCF fluorescence revealed a significant increase in ROS levels in a dose-dependent manner ( Figure 7B). The above results indicated that compound 9 could facilitate ROS accumulation in the MDA-MB-468 and 4T1 cells and revealed that compound 9 induced oxidative stress and mitochondrial dysfunction during the apoptosis of the MDA-MB-468 and 4T1 cells. DCF fluorescence demonstrated that at higher concentrations of treatment of compound 9 in the MDA-MB-468 and 4T1 cells through flow cytometry, the fluorescence became stronger which indicated that the ROS levels were higher ( Figure 7A). In detail, compound 9 of 30 µM in the MDA-MB-468 cells and 20 µM in the 4T1 cells significantly increased the ROS levels (p < 0.001) compared to the control group. In addition, the results from the fluorescence microscope were identical; DCF fluorescence revealed a significant increase in ROS levels in a dose-dependent manner ( Figure 7B). The above results indicated that compound 9 could facilitate ROS accumulation in the MDA-MB-468 and 4T1 cells and revealed that compound 9 induced oxidative stress and mitochondrial dysfunction during the apoptosis of the MDA-MB-468 and 4T1 cells. In summary, ten compounds were isolated from the rhizome of Belamcanda chinensis (L.) DC, including four compounds (1, 5, 7, and 8) first isolated from the genus Belamcanda Adans. nom. conserv. and six known compounds (2-4, 6, 9, and 10). Ten compounds had antiproliferative effects against five breast cancer cell lines (BT549, 4T1, MCF7, MDA-MB-231, and MDA-MB-468). Among them, compound 9 had the best inhibitory effect against the 4T1 and MDA-MB-468 cells. In addition, antiproliferative effects, apoptosis induction, In summary, ten compounds were isolated from the rhizome of Belamcanda chinensis (L.) DC, including four compounds (1, 5, 7, and 8) first isolated from the genus Belamcanda Adans. nom. conserv. and six known compounds (2-4, 6, 9, and 10). Ten compounds had antiproliferative effects against five breast cancer cell lines (BT549, 4T1, MCF7, MDA-MB-231, and MDA-MB-468). Among them, compound 9 had the best inhibitory effect against the 4T1 and MDA-MB-468 cells. In addition, antiproliferative effects, apoptosis induction, and potential molecular mechanisms of action were evaluated for the first time. It was demonstrated that compound 9 showed significant antiproliferative activity and inhibited metastasis in the MDA-MB-468 and 4T1 cells. Subsequently, Annexin V-FITC/PI showed an increase in apoptotic rates, and with Hoechst 33342 staining, significant changes in cell morphology were found after treatment with compound 9 of 10 µM to 30 µM in the MDA-MB-468 and 4T1 cells. As we know, depolarization of the mitochondrial membrane leads to the release of pro-apoptotic factors in mitochondria and the activation of apoptotic proteases and ultimately induces apoptosis. Excessive production of ROS leads to oxidative damage to important cellular molecules and structures, resulting in mitochondrial dysfunction and activation of endogenous apoptotic pathways and ultimately causing cell apoptosis [63]. As mentioned earlier, the changes in the mitochondrial membrane potential level and the ROS level revealed that compound 9 could induce apoptosis in MDA-MB-468 and 4T1 cells for the first time. In conclusion, all of the results revealed compound 9 may be a leading compound for triple-negative breast cancer to merit further study.

Separation and Purification
Belamcandae chinensis rhizoma was purchased from Heshun Tang Pharmacy in Shenzhen, China, in October 2020 and identified by Dr. Li Chenyang from Shenzhen University. Approximately 19 kg Belamcandae chinensis rhizoma was ground into a fine powder and extracted with 95% ethanol by refluxing extraction three times, each time for 2 h. The extraction solution was combined, the pressure was reduced, and it was evaporated to a certain volume, and then the crude extract was further fractionated through macroporous resin column chromatography and eluted successively with 45%, 75%, and 95% ethanol, which was brought to dryness under reduced pressure, yielding dried 45% (3.461 kg), 75% (350 g), and 95% (341 g) ethanol extract, respectively. The cytotoxicity assay of the three extracts ( Figure S1) indicated that the 75% ethanol extract showed the highest activity. Therefore, isolation for the active compounds from the 75% ethanol extract of Belamcandae chinensis rhizoma was carried out.

Cell Viability Assay
The cells were seeded in 96-well plates with 3 × 10 4 cells and 1 × 10 4 cells per well, incubated in a 37 • C incubator with 5% CO 2 added overnight. The cells were treated with compounds at different concentrations for 48 h in 5% CO 2 at 37 • C. The original medium was discarded, and each well was filled with 100 µL of culture medium containing 10% CCK-8 and incubated for 1 h at 37 • C in an incubator, and then the absorbance at 450 nm was detected with a microplate reader (POLARstar Omega, Washington, DC, USA).

Colony Formation
The cells were seeded in a 6-well plate (2 × 10 3 cells/well) and cultured overnight in 5% CO 2 at 37 • C for 7 days. When the colonies formed, they were treated with compounds at different concentrations, and then they were cultured in a 37 • C incubator with 5% CO 2 for 10 days. The fresh complete culture medium was replaced every other day. When the colonies were cultured to the naked eye, the colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution.

Cell Cycle Analysis
The cells were seeded in a 6-well plate and cultured overnight in 5% CO 2 at 37 • C. Then, the cells were treated with drugs of different concentrations for 48 h in 5% CO 2 at 37 • C. The original medium was discarded, and the cells were harvested and fixed by 70% ice-cold (v/v) ethanol overnight at 4 • C. After that, the fixed cells were centrifuged and washed with cold phosphate-buffered saline (PBS), and then, they were further resuspended in a solution of 0.5 mL propidium iodide (PI) and RNAase staining buffer for 30 min at 37 • C in the dark. The cells were determined by an Agilent NovoCyte Flow Cytometer, and the data were analyzed by NovoExpress software (version 1.6.1).

Wound-Healing Assay
The cells were seeded in a 6-well plate in a 37 • C incubator with 5% CO 2 , and after the cells reached confluency, a sterile pipette tip was used to draw parallel lines in each cell, and the cells were washed with PBS. In succession, a medium with 2% FBS was added with compounds at different concentrations, and a microscope was used to take pictures at 0 h, 24 h, and 48 h to analyze the results.

Transwell Assay
The cells were resuspended in 200 µL FBS-free medium into the upper chamber, and 600 µL complete medium with compounds at different concentrations was added into the lower chamber. After incubating for 48 h, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. The cells were photographed and counted under the microscope.

Annexin V-FITC/PI Double Staining Assay
Apoptosis was detected by an Annexin V-FITC/PI staining kit. The cells were seeded in a 6-well plate and cultured overnight in 5% CO 2 at 37 • C. Then, the cells were treated with drugs of different concentrations for 48 h in 5% CO 2 at 37 • C. The original medium was discarded, and the cells were collected and washed twice with cold PBS. The collected cells were dispersed with 100 µL 1 × binding buffer, and then, they were incubated with 5 µL Annexin V-FITC and 10 µL PI staining solution for 15 min at room temperature in the dark. The stained cells were detected by an Agilent NovoCyte Flow Cytometer, and the data were analyzed by NovoExpress software (version 1.6.1).

Hoechst 33342 Staining
The cells were seeded in a 6-well plate and cultured overnight in 5% CO 2 at 37 • C. After the cells were treated with compounds at different concentrations for 48 h in 5% CO 2 at 37 • C, the cells were washed twice with PBS and then stained with Hoechst 33342 fluorescent dye for 30 min at 37 • C in the dark. Then, using a fluorescence microscope, morphological changes in the nucleus were examined.

JC-1 Staining
Mitochondrial membrane potential (MMP) was determined by an MMP assay kit. Briefly, the cells were seeded in a 6-well plate and cultured overnight in 5% CO 2 at 37 • C. After the cells were treated with compounds of different concentrations, the medium was removed and washed with sterile PBS, and then, the cells were stained with a JC-1 fluorescent probe for 30 min at 37 • C in the dark. Thereafter, the cells were washed with 1 × JC-1 buffer solution three times, and the cells were pictured via fluorescence microscopy.

Measurement of Intracellular ROS Levels
The intracellular ROS levels were detected with an ROS assay kit. The cells were seeded in a 6-well plate and cultured overnight in 5% CO 2 at 37 • C. After the cells were treated with compounds at different concentrations, the medium was removed and washed with sterile PBS, and, on one hand, incubated with a DCFH-DA probe for 20 min at 37 • C in the dark, and then, the cells were washed with PBS three times, and the cells were pictured via fluorescence microscopy. On the other hand, the cells were collected, stained with a DCFH-DA probe, and ROS were determined by an Agilent NovoCyte Flow Cytometer, and the data were analyzed by NovoExpress software (version 1.6.1).

Statistical Analysis
All cell culture assays were performed in triplicate. The data were analyzed and processed by GraphPad Prism 9.0 software. The data were expressed in the form of the mean ± SD. Differences between the groups were compared with the statistical significance by a t-test. p < 0.05 represents a statistical difference.

Conclusions
In this study, four compounds (1, 5, 7, and 8) were first isolated from the genus Belamcanda Adans. nom. conserv., and six known compounds (2-4, 6, 9, and 10) were isolated from the rhizome of Belamcanda chinensis (L.) DC. The antiproliferative effects against five tumor cell lines (BT549, 4T1, MCF7, MDA-MB-231, and MDA-MB-468) suggested that compound 9 had the best inhibitory effect against the MDA-MB-468 and 4T1 cells. Subsequently, we found compound 9 could inhibit the proliferation and metastasis of MDA-MB-468 and 4T1 cells and induce cell cycle arrest in the G1 phase. Moreover, we found that compound 9 could downregulate mitochondrial membrane potential and produce excessive ROS to induce apoptosis of the MDA-MB-468 and 4T1 cells for the first time. In conclusion, our findings demonstrate that compound 9 may be a leading compound for triple-negative breast cancer to merit further study.