Hydroxypropyl-β-Cyclodextrin Complexes of Styryllactones Enhance the Anti-Tumor Effect in SW1116 Cell Line

Styryllactones, a class of compounds obtained from the genus Goniothalamus (Annonaceae), have demonstrated in vitro antitumor activity. However, the aqueous solubility of these compounds is poor. In this study, we identified the absolute configurations of the previously isolated compounds, which were first isolated in our laboratory, by single-crystal X-ray diffraction analysis using Cu Kα radiation. Subsequently, the antitumor activities of the compounds were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide staining in four tumor cell lines. The induced apoptosis activity of leiocarpin E-7ʹ-Monoacetate was studied by an annexin V fluorescein isothiocyanate/propidium iodide double-staining experiment, and the caspase activity was tested in the SW1116 cell line. The results demonstrated that the antitumor activities of cheliensisin A and goniodiol-7-monoacetate were limited by their poor water solubility. To address this issue, hydroxypropyl-β-cyclodextrin (HP-β-CD) complexes of the compounds were synthesized by the saturated aqueous method. The complexes were then analyzed using a differential scanning calorimeter. The IC50 of cheliensisin A was reduced by 45% and 58% against SW1116 and SMMC-7721 cell lines, respectively. Similarly, the IC50 of goniodiol-7-monoacetate was reduced by 55% and 34% against the two tumor cell lines, respectively. To further evaluate whether the styryllactones and complexes possessed selectivity against cancer cell lines and normal cell lines, toxicity against human normal cell line (HEK293T) was evaluated. The results demonstrated that the HP-β-CD complexes displayed more cytotoxicity than the respective pristine compounds against the HEK293T cell line. However, there existed a therapeutic window when the complexes were applied against cancer cell lines. In summary, the synthesis of several styryllactone compounds complexed with HP-β-CD was reported for the first time. These complexes could significantly enhance the cytotoxic effects of styryllactone compounds.


INTRODUCTION
Colon cancer is the third most common type of cancer worldwide in both men and women, and is associated with a high recurrence rate and increasing mortality rate (Lao and Grady, 2011;Purushotham et al., 2012;Altobelli et al., 2014;Siegel et al., 2014;Sunkara and Hebert, 2015). The existing treatment regimens for colon cancer include chemotherapy, radiotherapy, and surgical ablation. Among these, chemotherapy is the most common strategy (Wang et al., 2014b;Pohl and Schmiegel, 2016). However, the two major challenges for the effective treatment of colon cancer are adverse effects due to cancer chemotherapy and drug resistance (Kozovska et al., 2014;Lim et al., 2019). Hence, it is imperative to search for new chemotherapeutic agents that have better safety and efficacy profiles. In this context, the application of natural compounds is a promising approach (Sridhar et al., 2014;Levrier et al., 2015;Wang et al., 2016).
Studies have shown that some styryllactones possess potent cytotoxicity against human colon tumor cell lines. Ali et al. reported that goniothalamin exhibited the highest cytotoxic activity against HGC-27 cells among the different cell lines tested (HGC-27, MCF-7, PANC-1, HeLa) (Ali et al., 1997). Vendramini-Costa et al. demonstrated the importance of goniothalamin as a proapoptotic, and therapeutic agent for the treatment inflammatory bowel disease and emphasized its potential as a chemopreventive agent for colon cancer (Vendramini-Costa et al., 2016). Cheliensisin A, a novel styryllactone isolated from Goniothalamus cheliensis Hu, could trigger p53-mediated apoptosis, accompanied by dramatic inhibition of the anchorageindependent growth of HCT116 cells, thus highlighting its potential cancer therapeutic effect (Zhang et al., 2014).
In recent years, mechanisms related to the antitumor activity of styryllactone compounds have been reported. For example, goniothalamin induced the release of inflammatory cytokines by upregulating the B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)/Bcl-2, phosphorylate c-Jun N-terminal kinase (p-JNK1)/ JNK1, and p-p38/p38 ratios, which led to cleavage of poly (ADP-ribose) polymerase (PARP) and, finally resulted in apoptosis of the HT-29 cells (Vendramini-Costa et al., 2016). The cells were unable to grow without the BIRC 5 (Full name: the baculoviral inhibitor of apoptosis repeat-containing 5) protein.
While goniothalamin has demonstrated inhibitory action against transcription of the BIRC5 gene at the RNA level, thus subjecting NCI-H460 cells to DNA damage (Semprebon et al., 2014).
In this study, the absolute configurations of several styryllactone compounds, first isolated in our laboratory, were determined by single-crystal X-ray diffraction analysis using Cu Ka radiation. In addition, we evaluated the effect of complexation of sytryllactones with HP-b-CD on their antitumor activity. The styryllactones displayed enhanced antitumor activity when complexd with HP-b-CD.

Single-Crystal X-Ray Analysis
Data for diffraction intensity was obtained using a Bruker APEX-IICCD X-ray diffractometer (Bruker AXSGmbH, Karlsruhe, Germany) and graphite-monochromated Cu Ka radiation (l= 1.54178 Å). Cell refinement and data reduction were performed with Bruker SAINT (Bruker AXS, GmbH, Karlsruhe, Germany). The absorption correction was determined semi-empirically from equivalent compounds. The structures were determined via direct methods using SHELXS-97 (Institute of Inorganic Chemistry of Georg-August-Universität Göttingen, Gottingen, Germany). Non-hydrogen atoms were anisotropically refined with SHELXL-97 (Non-hydrogen atoms of leiocarpin B were anisotropically refined with SHELXL-2014). Hydrogen atoms were located by geometry and positioned on the related atoms during refinements, with a temperature factor.

Cell Culture and Assay
The human colon cancer SW1116 cell line, the human hepatocellular carcinoma SMMC-7721 cell line, the human gastric cancer SGC-7901 cell line, and the human hepatocellular carcinoma HepG2 cell line were kindly provided by Xiao-li Xu from the Cancer Center, Fudan University. The human embryonic kidney 293T (HEK293T) cell line was kindly donated by Professor You-hua Xie from Fudan University. These human cancer cell lines were cultured in DMEM medium or RPMI-1640 medium, whereas the HEK293T cell line was cultured in MEM medium. All of the cell lines were supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) under a humidified atmosphere of 5% CO 2 at 37°C using a CO 2 incubator (SANYO, Osaka, Japan). Cell count was assessed using the trypan blue dye exclusion method.
The antiproliferative effects of the treatments were evaluated using the MTT assay. Cells were seeded at a density of 5×10 3 cells/well in 96-well plates (Corning, NY, USA). After attachment, the culture media were replaced with various concentrations of chemicals for 24 h. Then, the media in 96well plates were incubated with MTT reagent (5 mg/ml) for 4 h at 37°C. Subsequently, the culture medium was discarded and 100 ml of DMSO was added to each well, to solubilize the formazan crystal formed. The absorbance of formazan crystal solution was determined at 570 nm on a Multiskan FC from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

Annexin V-FITC/PI Double Staining by Flow Cytometry
The growing cells were incubated in 24-well microplates (Corning, NY, USA) for 24 h. The cells were then treated with various concentrations of leiocarpin E-7′-monoacetate or taxol in humidified air with 5% CO 2 at 37°C. After 36 h of incubation, the culture medium was discarded, and the cells were collected. For the apoptosis analysis, cells were suspended with 1×binding buffer (1×10 6 cells/ml) and then labeled with annexin V-FITC/ PI, as per the manufacturer's instructions (Keygentec, Nanjing, Jiangsu, China). The analysis of the samples was performed by flow cytometry (Becton-Dickinson Bioscience, San Jose, CA, USA), and the acquired data was analyzed by the CellQuest software (Becton-Dickinson Bioscience, San Jose, CA, USA).

Caspase Activity
To evaluate the activity of caspases, cell lysates were prepared after their respective designated treatments. The incubation of the growing cells was carried out in 24-well microplates (Corning, NY, USA) for 24 h. The cells were then treated with different concentrations of leiocarpin E-7′-monoacetate under humidified air with 5% CO 2 at 37°C. After 8 h of incubation, the culture medium was discarded, the cells collected and washed twice with PBS. The mixture was then centrifuged at 2,000 rpm for 5 min. The PBS supernatant was discarded and the cells (concentration, 5×10 6 cells) were collected. To these cells, icecold lysis buffer (150~200 ml) was added. The mixture was placed on ice for 30 min, and then centrifuged (10,000 rpm, 1 min) at 4°C . The supernatant, containing lysed protein, was carefully aspirated and transferred to a new tube. The protein concentration was then measured in 2 ml of the supernatant using the Bradford method. The caspase assays were then performed in 96-well microtiter plates (Keygentec, Nanjing, Jiangsu, China) by incubating 10 ml of protein cell lysate per sample in 80 ml of reaction buffer (1% NP-40, 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, and 10% glycerol) containing 10 ml of caspase substrate (2 mM). Lysates were incubated at 37°C for 4 h. Measuremet was done at 405 nm on a Multiskan FC from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The detailed analysis procedure is described in the manufacturer's protocol (Keygentec, Nanjing, Jiangsu, China).

Preparation of HP-b-CD Complex
A fixed quantity of the compound was weighed and evenly dispersed in an aqueous solution of HP-b-CD (molecular ratio of 1:2). The dispersion was equilibrated for 24 h at room temperature, under constant stirring. The supernatant was then lyophilized using a Christ Alpha1-2 Ld10 Freeze Dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany) to obtain the inclusion complex in a dry powder form. The content of styryllactones in the complex was determined using an ultraviolet (UV) spectrophotometer (Shimadzu, Kyoto, Japan). When the complexes were used in the antitumor test, we first prepared a solution of the compound, and then performed full wavelength scanning. Subsequently, we selected the maximum absorption wavelength of the compound as the detection wavelength. A 20-mg quantity of the complex was accurately weighed and placed in a 10-ml volumetric flask. It was then dissolved in 0.1 mol/L hydrochloric acid-acetonitrilewater (1:1:2), and the volume was recorded. The absorbance was measured at the detection wavelength. We calculated the total amount of compound (W1) in the complex, according to the standard equation obtained with a compound solution prepared with 0.1 mol/L hydrochloric acid-acetonitrile-water (1:1:2). Another 20 mg of the same complex was accurately weighed, and dissolved in 0.1 mol/L hydrochloric acid-acetonitrile (1:1) and placed in an ultrasound machine for 10 min. Subsequently, it was filtered and the filtrate was used to measure the absorbance at the detection wavelength. We calculated the free compound content (W2), according to the standard equation obtained with a compound solution prepared with 0.1 mol/L hydrochloric acidacetonitrile (1:1). The difference between W1 and W2 represented the quantity of the compound that formed HP-b-CD complex in a 20-mg inclusion compound sample.

Differential Scanning Calorimetry
The thermal characteristics of the raw material, HP-b-CD, the physical mixtures, and the complexes were determined using a differential scanning calorimeter (DSC; NETZSCH DSC system), equipped with a computerized data station (TA-50WS/PC, Selb, Bavaria, Germany). Samples were accurately weighed in a crimped aluminum pan and heated under an inert atmosphere of nitrogen. An empty pan sealed in the same manner, was used as a reference. The scanning rate was 10°C/min, and the scanning temperature ranged between 30°C and 400°C.

Statistical Analysis
All data were expressed as means ± standard error of the mean (SEM) and were analyzed using two-tailed Student's t-tests. Statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). A value of p < 0.05 was considered statistically significant.

Styryllactones Inhibit the Proliferation of Tumor Cell Lines
The in vitro cytotoxic activity of styryllactones was evaluated in four human tumor cell lines by the MTT assay (Figure 3). VP-16 was chosen as the positive control, because it is one of the most widely used cancer chemotherapy agents to treat many kinds of cancers, and it could induce apoptosis of cancer cells by acting as a toposiomerase II inhibitor (Berger et al., 1996;Chiu et al., 2005). The results showed that cheliensisin A, goniodiol and goniodiol-7-monoacetate had no cytotoxic effect on the SGC-7901, SMMC-7721 and HepG2 cell lines since the IC 50 of these compounds were greater than 100 mM. In contrast, leiocarpin B, leiocarpin E, and leiocarpin E-7′-monoacetate showed excellent cytotoxicity against the SW1116 cell line, when their concentrations were 30 mM, the cell viability of SW1116 cells was significantly less (p < 0.01) than the normal group (cell viability in 0 mM). The highest cytotoxic effect against SW1116 cells was demonstrated by leiocarpin E-7′-monoacetate, while cheliensisin A and goniodiol-7-monoacetate showed relatively lower inhibitory effect (Table 1). Thus, it was concluded that the human colon cancer SW1116 cell line was much more sensitive to the cytotoxic effect of styryllactones.

Cytotoxic Effect of Styryllactones Against the Human Normal Cell Line HEK293T
The human embryonic kidney 293T (HEK293T) cell line is used as a normal human cell line in various biological experiments (Chen et al., 2016;Vemuri et al., 2019). In this study, we investigated the in vitro cytotoxic effect of styryllactones (concentration ranging from 0 and 100 mM) against HEK293T cells (Figure 4). The results showed that the IC 50 values of all the compounds against HEK293T cells were above 100 mM ( Table  1). This indicated that styryllactones did not show cytotoxicity against normal human cell line.

Leiocarpin E-7ʹ-Monoacetate Induces the Early Apoptosis of SW1116 Cells
In the in vitro cytotoxicity experiments, SW1116 cells were found to be sensitive to leiocarpin E-7′-monoacetate. To further evaluate the apoptotic activity of leiocarpin E-7′-monoacetate, the cells were treated with various concentrations of leiocarpin E-7′-monoacetate and then analyzed by flow cytometry. Taxol was chosen as the positive control, because it could stabilize microtubules, and subsequently cause cell apoptosis by arresting the cell cycle at G2/M (Ruden and Puri, 2013;Luo et al., 2015). The results showed that the early apoptosis rates were 2.6%, 21.8%, and 55.2% when the concentration of leiocarpin E-7′-monoacetate was 3, 10, and 30 mM, respectively ( Figure 5). In summary, the results indicated that leiocarpin E-7'-monoacetate was able to induce the apoptosis of SW1116 cells in a concentration-dependent manner.

Leiocarpin E-7ʹ-Monoacetate-Induced Apoptosis is Caspase-Dependent
To further examine the cytotoxicity induced by leiocarpin E-7′monoacetate, the activity of the apoptosis-associated protease was studied, using an enzyme activity assay kit. The results showed that different kinds of caspase proteases were activated as the concentration of leiocarpin E-7′-monoacetate was increased ( Figure 6). When the concentration was 3 mM, the ration of  OD sample to OD negative was not greater than 1, as it indicated that the caspase enzymes were not activated. However, when the concentration was increased to 10 and 30 mM, the ration was greater than 1, as it demonstrated that the activation of caspase enzymes increased significantly.

Identification of Complexes by Differential Scanning Calorimetry
In the cytotoxicity experiments, cheliensisin A and gonidiol-7-monoacetate showed no effects on tumor growth. This could be attributed to the poor water solubility of these compounds.
To improve the solubility, the styryllactones HP-b-CD complexes were synthesized via the saturated aqueous solution method. A DSC analysis was then carried out for HP-b-CD, styryllactone, a styryllactone/HP-b-CD physical mixture, and the styryllactone/HP-b-CD complex (Figure 7). The raw cheliensisin A ( Figure 7A) had a sharp endothermic peak and a sharp exothermic peak near 150°C and 300°C, respectively. HP-b-CD had a broad endotherm near 350°C, which was also present in the mixture. However, in the spectrum of the complex, the characteristic peak of cheliensisin A disappeared. This indicated that the compound penetrated into the cyclodextrin cavity and replaced the water molecules.
The raw goniodiol-7-monostearate monomer ( Figure 7B) had a sharp endothermic peak at 150°C, which were also present in the mixture. The DSC curves of the complex showed that the characteristic peaks of the goniodiol-7monostearate monomer disappeared, which confirmed the formation of the styryllactone complex with HP-b-CD.

Increased Cytotoxicity Activity of the Complex
To examine whether complexation of styryllactone with HP-b-CD resulted in an enhancement of antitumor activity against  different human cell lines, cytotoxicity experiments were evaluated in SMMC-7721 and SW1116 cell lines, using the MTT assay ( Figure 8).
The results showed that the styryllactone/HP-b-CD complexes were significantly more cytotoxic than their respective pristine form (P < 0.01), while HP-b-CD did not show any inhibition of the growth of the two cell lines ( Table 2). The IC 50 of cheliensisin A and goniodiol-7-monoacetate were reduced by 45% and 55%, respectively against the SW1116 cell line. Similarly, the IC 50 values of the two compounds were reduced by 58% and 34%, respectively against the SMMC-7721 cell line.

Cytotoxicity Activity Against HEK293T Cells is Increased by the Complex
Based on the results, it was observed that the complexes demonstrated enhanced cytotoxic effect against tumer cell lines, when compared with the styryllactone compounds. To study whether there is obviously enhanced cytotoxicity of complex in normal cell lines, the human normal cell line HEK293T was treated with styryllactone HP-b-CD complexes. Figure 9 suggested that the complexes showed significantly higher cytotoxic activity than the compounds against HEK293T cell line (P < 0.01). For example, the IC 50 of cheliensisin A complex and goniodiol-7-monoacetate complex were 98.46 ± 3.00 and 35.02 ± 0.63 mM, respectively ( Table 2). When the cells were treated with the pristine forms of cheliensisin A and goniodiol-7-monoacetate, the IC 50 values obtained were beyond 200 mM. In addition, HP-b-CD did not show any inhibitory activity against the HEK293T cell line (Figure 9).
In mammalian cells, there are two major apoptotic pathways: the first one involves a signal from the mitochondria, while the second relies on signal transduction through death receptors. Studies have shown that caspase-8 is activated through a death receptor-mediated pathway and cleavage of caspase-9 plays a key role in mitochondria apoptotic pathway. Caspase-3 is the key executive molecule of the apoptotic signal (Dodson et al., 2013;Vendramini-Costa et al., 2016). We observed the occurrence of apoptosis at the cellular level. Subsequently, we investigated whether the activity of caspase enzymes was affected owing to the intervention of leocarpin E-7'-monoacetate at the enzyme level. It was observed that caspase-3, -8 and -9 showed activation at different concentrations of leocarpin E-7'-monoacetate ( Figure 6). When the concentration of leocarpin E-7'monoacetate was 3 mM, in the caspase activity test the ration of OD sample to OD negative was not greater than 1, while the apoptosis rate is low. When the concentration was increase to 10 and 30 mM, the apoptosis rate also increased.
However, cheliensisin A, goniodiol, and goniodiol-7monoacetate showed poor in vitro activity. The application of styryllactones as antitumor agent has been strongly impeded owing to their poor water solubility (Zhao et al., 2008;Deng et al., 2011). In a study by Zhao et al, the solubility of cheliensisin A was improved by formulating it as a lyophilized submicron emulsion intravenous injection (Zhao et al., 2008). However, the process is complicated and expensive. In this study, we prepared inclusion complexes of styryllactones using HP-b-CD and evaluated the cytotoxic activity of the complex.
HP-b-CD is commonly used to enhance the water solubility of poorly soluble compounds. It contains a hydrophilic exterior surface and a nonpolar interior cavity. This structure allows HPb-CD to act as a carrier that can encapsulate a poorly watersoluble compound in the internal area, thereby increasing the solubility of the compound (Kaur et al., 2014;  2014a). In the pharmaceutical industry, HP-b-CD is used to increase drug stability, improve bioavailability, and reduce side effects owing to its low surface activity, low hemolytic activity, and lack of muscle irritation. Thus, it is widely used as an injection solubilizer and pharmaceutical excipient (Kryjewski et al., 2015).
Differential Scanning Calorimetry (DSC) is a suitable thermal analysis technique used in the pharmaceutical industry for determining the purity, polymorphic forms, and melting point of a sample (Demetzos, 2008). In addition, DSC can provide detailed information about both the physical and energetic properties of a substance (Green et al., 2020). The results showed that apart from the characteristic peaks of styryllactones and HP-b-CD, no other endothermic or exothermic peak was observed (Figure 7). In addition, cyclodextrin is a cyclic oligosaccharide composed of covalently-linked glucopyranose rings, which can assist in increasing the solubility of hydrophobic drugs by forming water-soluble inclusions (Brewster and Loftsson, 2007). HP-b-CD is a chemically modified derivate of cyclodextrin that has a higher solubility in water and can be safely used as a complexing and solubilizing excipient in various drug administration routes (Peeters et al., 2002). For example, Al-Qubaisi et al. developed an inclusion complex of thymoquinone and HP-b-CD in order to improve solubility and bioactivity of thymoquinone. In their work, they proved that the entire thymoquinone molecule was entrapped in the HP-b-CD cavity and that the molecule was not degraded by the complexation (Al-Qubaisi et al., 2019). As a result, we demonstrated that the cyclodextrin complexation did not degrade the molecular structure.
It was observed that the antitumor effect of styryllactones complexed with HP-b-CD was significantly enhanced. From the experiments, the IC 50 of the cheliensisin A complex was 51.44 ± 4.78 mM and 46.91 ± 1.60 mM against SW1116 cells and SMMC-7721 cells, respectively. The IC 50 of goniodiol-7-monoacetate complex was 46.48 ± 3.25 mM and 75.76 ± 4.19 mM, whereas the IC 50 of the cheliensisin A monomer was 93.18 ± 0.78 mM and 110.90 ± 2.41 mM against SW1116 cells and SMMC-7721 cells, respectively. The IC 50 of the goniodiol-7-monoacetate monomer was 102.14 ± 4.15 mM and 115.54 ± 3.97 mM, respectively ( Table  2). HP-b-CD had no inhibitory effect on the growth of the two cell lines (Figure 8). The cytotoxicity of styryllactones and their complex was also evaluated in the human normal cell line HEK293T. It was observed that the HP-b-CD complexes showed greater cytotoxic activity than the styryllactone compounds against HEK293T cell line. This suggested that the complexes could also inhibit the growth of normal cell line. The IC 50 of cheliensisin A complex and goniodiol-7-monoacetate complex were 98.46 ± 3.00 and 35.02 ± 0.63 mM, respectively ( Table 2). Thus, there was a dose safety window when cheliensisin A complexes were treated against SW1116 cell line or SMMC-7721 cell line. In addition, there was a dose safety window when goniodiol-7-monoacetate complexes were treated against SW1116 cell line. However, the dose safety window was narrower when goniodiol-7-monoacetate complexes were treated against SMMC-7721 cell line. In this study, we focused on investigation of the method to enhance the cytotoxicity activity of styryllactones. HP-b-CD is known to increase the solubility of poorly water-soluble compounds (Kaur et al., 2014;Wang et al., 2014a). Thus, it was assumed that the enhanced antitumor effects of the complexes were partly because of the improved water solubility. The results from this study show that styryllactones have the potential to be developed as antitumor compounds. Next, we plan to obtain enough compounds, either by extraction from plants or chemical synthesis and then explore better compound structures based on these styryllactones. Their HP-b-CD complex will be developed and cytotoxicity will be studied. Subsequently, we intend to investigate the anticancer effect in the animal model using the styryllactones inclusion complex, and conduct efficient test methods including radiolabeling test and permeability test, to illustrate the exact mechanism underlying the improved potency of complexes toward tumor cell lines.
This paper described the identification of the absolute configurations of several styryllactones by single-crystal X-ray diffraction analysis using Cu Ka radiation. We synthesized styryllactones complexes with HP-b-CD for the first time. The in vitro antitumor experiments showed that the inhibitory activity of these complexes was greater than the respective pristine form of the compounds. These results suggest that the water solubility of styryllactones can be improved by complexation of styryllactones with HP-b-CD, which in turn, can enhance the antitumor activity of these compounds.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
RM and QM contributed to the conception and design of the study. RM performed the experiments and wrote the first draft of the manuscript. J-TC assisted in the performance of experiments. X-LX donated the cells in the research. QM and X-YJ revised the manuscript. QM gave final approval of the version to be submitted. All authors read and approved the final manuscript.