The Inhibitory Effects and Cytotoxic Activities of the Stem Extract of Nepenthes miranda against Single-Stranded DNA-Binding Protein and Oral Carcinoma Cells

The carnivorous pitcher plants of the genus Nepenthes exhibit many ethnobotanical uses, including treatments of stomachache and fever. In this study, we prepared different extracts from the pitcher, stem, and leaf extracts of Nepenthes miranda obtained using 100% methanol and analyzed their inhibitory effects on recombinant single-stranded DNA-binding protein (SSB) from Klebsiella pneumoniae (KpSSB). SSB is essential for DNA replication and cell survival and thus an attractive target for potential antipathogen chemotherapy. Different extracts prepared from Sinningia bullata, a tuberous member of the flowering plant family Gesneriaceae, were also used to investigate anti-KpSSB properties. Among these extracts, the stem extract of N. miranda exhibited the highest anti-KpSSB activity with an IC50 value of 15.0 ± 1.8 μg/mL. The cytotoxic effects of the stem extract of N. miranda on the survival and apoptosis of the cancer cell lines Ca9-22 gingival carcinoma, CAL27 oral adenosquamous carcinoma, PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were also demonstrated and compared. Based on collective data, the cytotoxic activities of the stem extract at a concentration of 20 μg/mL followed the order Ca9-22 > CAL27 > PC9 > 4T1 > B16F10 cells. The stem extract of N. miranda at a concentration of 40 μg/mL completely inhibited Ca9-22 cell migration and proliferation. In addition, incubation with this extract at a concentration of 20 μg/mL boosted the distribution of the G2 phase from 7.9% to 29.2% in the Ca9-22 cells; in other words, the stem extract might suppress Ca9-22 cell proliferation by inducing G2 cell cycle arrest. Through gas chromatography–mass spectrometry, the 16 most abundant compounds in the stem extract of N. miranda were tentatively identified. The 10 most abundant compounds in the stem extract of N. miranda were used for docking analysis, and their docking scores were compared. The binding capacity of these compounds was in the order sitosterol > hexadecanoic acid > oleic acid > plumbagin > 2-ethyl-3-methylnaphtho[2,3-b]thiophene-4,9-dione > methyl α-d-galactopyranoside > 3-methoxycatechol > catechol > pyrogallol > hydroxyhydroquinone; thus, sitosterol might exhibit the greatest inhibitory capacity against KpSSB among the selected compounds. Overall, these results may indicate the pharmacological potential of N. miranda for further therapeutic applications.


Introduction
Single-stranded DNA-binding proteins (SSBs) are essential components in DNA metabolic processes, including replication, repair and recombination in both prokaryotes and eukaryotes [1,2]. Single-stranded DNA (ssDNA) is fragile and susceptible, and, thus, it is needed to be protected by SSB from nucleases and chemical attacks. SSB also functions to maintain the transient unwinding of duplex DNA in the single-stranded Many plant extracts have been known to help heal human infections due to their secondary metabolite content [25]. DNA replication is one of the most basic biological functions and, thus, SSB, an essential DNA replication protein, should be a prime target in antibiotic development [26]. Recently, we found that the flavonol myricetin can bind to and inhibit P. aeruginosa SSB (PaSSB) [27][28][29]. Our complexed crystal structure showed that the binding site of the inhibitor myricetin [29] overlapped with the ssDNA-binding sites of PaSSB [29][30][31][32]. The non-inhibitor quercetin, a myricetin analogue, can also bind to PaSSB; however, quercetin cannot inhibit PaSSB [27]. In this study, we found that myricetin and quercetin cannot inhibit KpSSB. We then screened for possible KpSSB inhibitor(s) from plant extracts. We found that the stem extract of N. miranda exhibited significant anti-KpSSB activity. The chemical composition of the stem extracts of N. miranda was analyzed via gas chromatography-mass spectrometry (GC-MS). To elucidate the possible inhibition mode, the 10 most abundant compounds in the stem extract of N. miranda were used for docking analysis of KpSSB. Further studies should focus on testing these active compounds for the development of drugs against KpSSB and Ca9-22 gingival carcinoma and to determine whether the stem extract of N. miranda can be used as an alternative treatment.

Binding of KpSSB to ssDNA
Since SSB is required for cell survival, it may be a suitable target for potential antipathogen chemotherapy. Before exploring inhibitor(s) against KpSSB, the binding of KpSSB to ssDNA of different lengths with different protein concentrations was studied using electrophoretic mobility shift analysis (EMSA) [33,34]. The expected result of EMSA for detecting the distinct protein-DNA complex(es) [35] is that when the length of the nucleotides is sufficient for the binding of SSB, the electrophoretic mobility of the SSB-DNA complex will be lower than that of the protein-unbound DNA. Different deoxythymidine (dT) homopolymers were used in this study. These ssDNAs were biotinylated at the 5 terminal and incubated with purified KpSSB at different concentrations ( Figure 1). A streptavidin-horseradish peroxidase conjugate was used to detect these biotin-labeled ssDNAs and their complexes [27,28]. When we incubated KpSSB with a 20-mer dT (dT20), no band shift was observed; that is, KpSSB could not form a stable complex with this homopolymer ( Figure 1A). We then tried to use longer ssDNA homopolymers, dT30 ( Figure 1B) and dT35 ( Figure 1C), for the binding of KpSSB. In contrast to dT20, these longer dT homopolymers produced a very significant band shift (C, complex). These results indicate that KpSSB is capable of forming a stable complex with dT30 and dT35. According to the titration curves of EMSA, the midpoint values for input ssDNA-binding ([Protein] 50 ) were quantified. The binding constants of KpSSB to dT30 and dT35 were calculated to be 155 ± 20 and 117 ± 18 nM, respectively (Table 1).   Values show mean standard deviation of at least three independent experiments.

The Flavonols Myricetin, Quercetin, Kaempferol, and Galangin Did Not Inhibit KpSSB
Previously, we found that the flavonol myricetin can bind to [29] and inhibit PaSSB [27]. The complexed crystal structure of PaSSB with myricetin revealed where the binding occurs, and we investigated the possible inhibition mode. Residues Lys7, Glu80, Ile105, Asn106, Gly107, and Asn108 in PaSSB were involved in myricetin binding (PDB entry 5YUN), of which Lys7 and Glu80 in PaSSB also interacted with ssDNA (PDB entry 6IRQ) [30,31]. Given that Lys7 and Glu80 are conserved in KpSSB (Lys8 and Glu81; see below), myricetin might also compete with ssDNA for binding sites and inhibit KpSSB. To assess

The Flavonols Myricetin, Quercetin, Kaempferol, and Galangin Did Not Inhibit KpSSB
Previously, we found that the flavonol myricetin can bind to [29] and inhibit PaSSB [27]. The complexed crystal structure of PaSSB with myricetin revealed where the binding occurs, and we investigated the possible inhibition mode. Residues Lys7, Glu80, Ile105, Asn106, Gly107, and Asn108 in PaSSB were involved in myricetin binding (PDB entry 5YUN), of which Lys7 and Glu80 in PaSSB also interacted with ssDNA (PDB entry 6IRQ) [30,31]. Given that Lys7 and Glu80 are conserved in KpSSB (Lys8 and Glu81; see below), myricetin might also compete with ssDNA for binding sites and inhibit KpSSB. To assess whether myricetin inhibited the binding activity of KpSSB, myricetin (0-300 µM) was included in the binding assay ( Figure 3A). A concentration of 625 nM KpSSB, sufficient to reach 100% binding of the dT30 ssDNA (Figure 1), was used for this inhibition test. Unlike the result of PaSSB, myricetin did not inhibit KpSSB binding to dT30. Other myricetin analogues, i.e., quercetin ( Figure 3B), kaempferol ( Figure 3C), and galangin ( Figure 3D) bearing different numbers of hydroxyl substituents on the aromatic rings, were also used for the inhibition test of KpSSB. Each of these flavonols (0-300 µM) was included in the binding assay. We found that these flavonols did not inhibit the binding of KpSSB to dT30 either. Thus, myricetin is an inhibitor against PaSSB but not against KpSSB.
cluded in the binding assay ( Figure 3A). A concentration of 625 nM KpSSB, sufficient to reach 100% binding of the dT30 ssDNA (Figure 1), was used for this inhibition test. Unlike the result of PaSSB, myricetin did not inhibit KpSSB binding to dT30. Other myricetin analogues, i.e., quercetin ( Figure 3B), kaempferol ( Figure 3C), and galangin ( Figure 3D) bearing different numbers of hydroxyl substituents on the aromatic rings, were also used for the inhibition test of KpSSB. Each of these flavonols (0-300 μM) was included in the binding assay. We found that these flavonols did not inhibit the binding of KpSSB to dT30 either. Thus, myricetin is an inhibitor against PaSSB but not against KpSSB.

Inhibition of KpSSB by Plant Extracts
None of the flavonols used was judged to be an inhibitor against KpSSB ( Figure 3). We screened different plant extracts of N. miranda and Sinningia bullata for a KpSSB inhibitor. S. bullata is a tuberous member of the flowering plant family Gesneriaceae [36,37]. Leaf, stem, and pitcher of N. miranda and leaf, stem, and tuber of S. bullata were extracted using 100% methanol to assess whether these extracts inhibit KpSSB activity ( Figure 4). Each of these extracts (at concentrations of 7.8-1000 μg/mL) was included in the binding assay. According to the results from EMSA, the inhibition ability of these extracts was in the following order: the stem extract of N. miranda ( Figure 4A) > the pitcher extract of N. miranda ( Figure 4B) > the leaf extract of N. miranda ( Figure 4C) > the leaf extract of S. bullata ( Figure 4D) > the stem extract of S. bullata ( Figure 4E) > the tuber extract of S. bullata ( Figure  4F). Even at 1000 μg/mL, the tuber extract of S. bullata did not influence the binding of KpSSB to ssDNA ( Figure 4G). The stem extract of N. miranda exhibited the highest inhibition effect against KpSSB among these extracts, with an IC50 value of 15.0 ± 1.8 μg/mL (Table 2). Accordingly, certain compounds in the stem extract of N. miranda obtained using 100% methanol could be potential KpSSB inhibitors.

Inhibition of KpSSB by Plant Extracts
None of the flavonols used was judged to be an inhibitor against KpSSB ( Figure 3). We screened different plant extracts of N. miranda and Sinningia bullata for a KpSSB inhibitor. S. bullata is a tuberous member of the flowering plant family Gesneriaceae [36,37]. Leaf, stem, and pitcher of N. miranda and leaf, stem, and tuber of S. bullata were extracted using 100% methanol to assess whether these extracts inhibit KpSSB activity ( Figure 4). Each of these extracts (at concentrations of 7.8-1000 µg/mL) was included in the binding assay. According to the results from EMSA, the inhibition ability of these extracts was in the following order: the stem extract of N. miranda ( Figure 4A) > the pitcher extract of N. miranda ( Figure 4B) > the leaf extract of N. miranda ( Figure 4C) > the leaf extract of S. bullata ( Figure 4D) > the stem extract of S. bullata ( Figure 4E) > the tuber extract of S. bullata ( Figure 4F). Even at 1000 µg/mL, the tuber extract of S. bullata did not influence the binding of KpSSB to ssDNA ( Figure 4G). The stem extract of N. miranda exhibited the highest inhibition effect against KpSSB among these extracts, with an IC 50 value of 15.0 ± 1.8 µg/mL (Table 2). Accordingly, certain compounds in the stem extract of N. miranda obtained using 100% methanol could be potential KpSSB inhibitors.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
Owing to its high inhibitory capability toward KpSSB, the stem extract of N. miranda was chosen for GC-MS analysis to determine the compounds present. The compounds detected in this extract were tentatively identified by matching generated spectra with the NIST 2011 and Wiley 10th edition mass spectral libraries. The 16 most abundant compounds (>0.6%) were as follows: plumbagin, sitosterol, hydroxyhydroquinone, pyrogallol, hexadecanoic acid, oleic acid, 2-ethyl-3-methylnaphtho[2,3-b]thiophene-4,9-dione, catechol, 3-methoxycatechol, methyl α-D-galactopyranoside, 2-phenylethanol, epicurzerenone, stigmasterol, methyl palmitate, 13-docosenamide, and stigmasta-3,5-diene ( Figure 5).   Given its high inhibitory capability against the SSB, the stem extract of N. miranda was also used to test its cytotoxic activities. Initially, a trypan blue staining assay was used to estimate the cytotoxic effects of this extract ( Figure 6A). The trypan blue staining assay is based on the principle that live cells possess intact cell membranes that exclude certain dyes, e.g., trypan, whereas dead cells do not [38]. Accordingly, this assay allows for the direct identification and enumeration of live (unstained) and dead (blue) cells in a given population. The cancer cell lines Ca9-22 gingival carcinoma, CAL27 oral adenosquamous carcinoma, PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were used to evaluate the anticancer activities of the stem extract of N. miranda. The monolayers prepared in 96-well microtitration plates for these cancer cells were individually inoculated with this extract at different concentrations (10,20,40, and 80 µg/mL) per well. The death rate of cells treated with the stem extract of N. miranda was estimated. The cancer cells incubated with 80 µg/mL of this extract were completely killed ( Figure 6B). Incubation with 20 µg/mL of the stem extract of N. miranda caused the deaths of Ca9-22, CAL27, PC9, 4T1, and B16F10 cells at rates of 60, 30, 5, 4, and 3%, respectively. The cytotoxic activities followed the order Ca9-22 > CAL27 > PC9, 4T1, and B16F10 cells. Thus, the stem extract of N. miranda was far more effective against human OSCC cells, i.e., Ca9-22 and CAL27 cells, than against PC9, 4T1, and B16F10 cells.

Dose-Dependent Cytotoxic Effects of the Stem Extract of N. miranda on the Survival of Ca9-22, CAL27, PC9, 4T1, and B16F10 Cells
Given its high inhibitory capability against the SSB, the stem extract of N. miranda was also used to test its cytotoxic activities. Initially, a trypan blue staining assay was used to estimate the cytotoxic effects of this extract ( Figure 6A). The trypan blue staining assay is based on the principle that live cells possess intact cell membranes that exclude certain dyes, e.g., trypan, whereas dead cells do not [38]. Accordingly, this assay allows for the direct identification and enumeration of live (unstained) and dead (blue) cells in a given population. The cancer cell lines Ca9-22 gingival carcinoma, CAL27 oral adenosquamous carcinoma, PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were used to evaluate the anticancer activities of the stem extract of N. miranda. The monolayers prepared in 96-well microtitration plates for these cancer cells were individually inoculated with this extract at different concentrations (10,20,40, and 80 μg/mL) per well. The death rate of cells treated with the stem extract of N. miranda was estimated. The cancer cells incubated with 80 μg/mL of this extract were completely killed ( Figure 6B). Incubation with 20 μg/mL of the stem extract of N. miranda caused the deaths of Ca9-22, CAL27, PC9, 4T1, and B16F10 cells at rates of 60, 30, 5, 4, and 3%, respectively. The cytotoxic activities followed the order Ca9-22 > CAL27 > PC9, 4T1, and B16F10 cells. Thus, the stem extract of N. miranda was far more effective against human OSCC cells, i.e., Ca9-22 and CAL27 cells, than against PC9, 4T1, and B16F10 cells. Whether the stem extract of N. miranda could induce apoptosis of Ca9-22, CAL27, PC9, 4T1, and B16F10 cells was also investigated ( Figure 7A). The apoptosis induced by this extract in these cancer cells was estimated by using the Hoechst staining assay. Hoechst 33342 is membrane-permeable and detects all nucleated cells [39]. Through the Hoechst staining assay, we found that the stem extract of N. miranda, at a concentration of 80 μg/mL, could induce apoptosis ( Figure 7B), with 100% DNA fragmentation identified in these cancer cells. Incubation with 20 μg/mL of the stem extract of N. miranda induced apoptosis in Ca9-22, CAL27, PC9, 4T1, and B16F10 cells at the rates of 65, 33, 10, 8, 7%,  extract in these cancer cells was estimated by using the Hoechst staining assay. Hoechst 33342 is membrane-permeable and detects all nucleated cells [39]. Through the Hoechst staining assay, we found that the stem extract of N. miranda, at a concentration of 80 µg/mL, could induce apoptosis ( Figure 7B), with 100% DNA fragmentation identified in these cancer cells. Incubation with 20 µg/mL of the stem extract of N. miranda induced apoptosis in Ca9-22, CAL27, PC9, 4T1, and B16F10 cells at the rates of 65, 33, 10, 8, 7%, respectively. Similar to results obtained from the trypan blue staining assay, the apoptosis-inducing activities also followed the order Ca9-22 > CAL27 > PC9, 4T1, and B16F10 cells.

The Stem Extract of N. miranda Exhibited Cytotoxicity against Ca9-22 Cell Migration and Proliferation
We found that the stem extract of N. miranda also exhibited cytotoxicity against Ca9-22 cell migration and proliferation. The migration and proliferation of Ca9-22 cells were analyzed using wound-healing and clonogenic formation assays, respectively ( Figure  8A). Results show that the stem extract of N. miranda strongly inhibited the migration and proliferation of Ca9-22 cells. After 24 h of incubation, the stem extract of N. miranda at concentrations of 10, 20, 40, and 80 μg/mL reduced Ca9-22 cell migration by 28%, 68%, 100%, and 100%, respectively ( Figure 8B). A clonogenic formation assay further revealed that pretreatment with the stem extract of N. miranda at concentrations of 10, 20, 40, and 80 μg/mL suppressed the proliferation and colony formation of Ca9-22 cell by 17%, 66%, 99%, and 100%, respectively ( Figure 8C). Accordingly, the stem extract of N. miranda at a concentration of 40 μg/mL completely inhibited Ca9-22 cell migration and proliferation.

The Stem Extract of N. miranda Exhibited Cytotoxicity against Ca9-22 Cell Migration and Proliferation
We found that the stem extract of N. miranda also exhibited cytotoxicity against Ca9-22 cell migration and proliferation. The migration and proliferation of Ca9-22 cells were analyzed using wound-healing and clonogenic formation assays, respectively ( Figure 8A). Results show that the stem extract of N. miranda strongly inhibited the migration and proliferation of Ca9-22 cells. After 24 h of incubation, the stem extract of N. miranda at concentrations of 10, 20, 40, and 80 µg/mL reduced Ca9-22 cell migration by 28%, 68%, 100%, and 100%, respectively ( Figure 8B). A clonogenic formation assay further revealed that pretreatment with the stem extract of N. miranda at concentrations of 10, 20, 40, and 80 µg/mL suppressed the proliferation and colony formation of Ca9-22 cell by 17%, 66%, 99%, and 100%, respectively ( Figure 8C). Accordingly, the stem extract of N. miranda at a concentration of 40 µg/mL completely inhibited Ca9-22 cell migration and proliferation.

The Stem Extract of N. miranda Suppressed Carcinoma Cell Proliferation by Inducing G2 Cell-Cycle Arrest
Due to having the highest activities, Ca9-22 cells were chosen for the analysis of the cell cycle progression induced by the stem extract of N. miranda. The cell cycle impact was examined using flow cytometry (Figure 9). Incubation with the stem extract of N. miranda at concentrations of 10 and 20 μg/mL boosted the distribution of the G2 phase from 7.9% to 16.6% and 29.2% in the Ca9-22 cells, respectively. Therefore, the stem extract of N. miranda might suppress carcinoma cell proliferation by inducing G2 cell cycle arrest.

Molecular Docking
Our GC-MS results show that at least 16 compounds were detected in the stem extract of N. miranda ( Figure 5), and, thus, certain compounds in this extract might be responsible for the inhibition of KpSSB. Recently, we solved the crystal structure of KpSSB [33]; therefore, this structure is available for the study of molecular docking (PDB ID 7F2N). To predict the binding site(s), we elucidated the compound's mode of binding to KpSSB and calculated the binding energies using the MOE (molecular operating environment) Dock tool ( Figure 10). The 10 most abundant compounds in the stem extract of N. miranda were used for docking analysis. According to the docking score (the S score), the binding affinities were predicted for the KpSSB-ligand complexes with all possible binding geometries. Based on the S scores (Table 3), the binding capacity of these compounds was in the following order: sitosterol ( Figure 10A) > hexadecanoic acid ( Figure 10B) > oleic acid ( Figure  10C) > plumbagin ( Figure 10D) > 2-ethyl-3-methylnaphtho[2,3-b]thiophene-4,9-dione ( Figure 10E) > methyl α-D-galactopyranoside ( Figure 10F) > 3-methoxycatechol ( Figure  10G) > catechol ( Figure 10H) > pyrogallol ( Figure 10I) > hydroxyhydroquinone ( Figure  10J). As it had the highest S score, sitosterol might exhibit the greatest binding affinity toward KpSSB among the selected compounds. According to the modeled structure of the KpSSB-ssDNA complex ( Figure 10K), it is likely all these compounds occupy the ssDNA-

Molecular Docking
Our GC-MS results show that at least 16 compounds were detected in the stem extract of N. miranda ( Figure 5), and, thus, certain compounds in this extract might be responsible for the inhibition of KpSSB. Recently, we solved the crystal structure of KpSSB [33]; therefore, this structure is available for the study of molecular docking (PDB ID 7F2N). To predict the binding site(s), we elucidated the compound's mode of binding to KpSSB and calculated the binding energies using the MOE (molecular operating environment) Dock tool ( Figure 10). The 10 most abundant compounds in the stem extract of N. miranda were used for docking analysis. According to the docking score (the S score), the binding affinities were predicted for the KpSSB-ligand complexes with all possible binding geometries. Based on the S scores (Table 3), the binding capacity of these compounds was in the following order: sitosterol ( Figure 10A) > hexadecanoic acid ( Figure 10B) > oleic acid ( Figure 10C) > plumbagin ( Figure 10D) > 2-ethyl-3-methylnaphtho[2,3-b]thiophene-4,9-dione ( Figure 10E) > methyl α-D-galactopyranoside ( Figure 10F) > 3-methoxycatechol ( Figure 10G) > catechol ( Figure 10H) > pyrogallol ( Figure 10I) > hydroxyhydroquinone ( Figure 10J). As it had the highest S score, sitosterol might exhibit the greatest binding affinity toward KpSSB among the selected compounds. According to the modeled structure of the KpSSB-ssDNA complex ( Figure 10K), it is likely all these compounds occupy the ssDNA-binding sites of KpSSB ( Figure 10L). The KpSSB inhibitory capacity of the stem extract of N. miranda might derive from the co-effects of some of these compounds.

Discussion
Carnivorous plants are polyphyletic and represented by approximately 850 carnivorous plant species [40]. Carnivorous syndromes have evolved independently in approximately 10 lineages of flowering plants [41,42]. Carnivorous plants such as N. miranda, which was used in this study, possess specialized traps derived from leaves that are able to attract and catch small animals such as insects and retain them in these pitchers. The captured organisms killed, digested, and assimilated by the plant are a source of additional nutrients for plant growth, reproduction, and adaptation to nutrient-poor habitats. In this study, we found that the stem extract of N. miranda is capable of inhibiting the activity of KpSSB ( Figure 4) and suppressing the survival, proliferation, and migration abilities of Ca9-22 carcinoma cells (Figures 6 and 8). The stem extract of N. miranda also strongly induced apoptosis with DNA fragmentation in the Ca9-22 cells (Figure 7). The flow cytometry results indicate that this extract might suppress Ca9-22 cell proliferation by inducing G2 cell cycle arrest (Figure 9). Given that the G2 cell cycle checkpoint is a critical genome guardian of tumor cells [43] and G2 checkpoint abrogation has been considered to be a promising therapeutic anticancer target [43,44], the cellular signaling pathways that trigger this G2 arrest in Ca9-22 cells caused by the stem extract of N. miranda should be further experimentally investigated.
Nepenthes has long been used in folk medicine around the world for the treatment of various disorders, such as stomachache and fever [17]. The extracts of Nepenthes, due to their (long-time) ethnomedicinal uses, are safe as pharmaceuticals and should have fewer side effects for human use. Thus, more therapeutic applications should be discovered and developed. Recent investigations further confirmed that some Nepenthes extracts have significant anticancer and antibacterial activities [16,18,20,45,46]. In this study, we identified SSB as one of the molecular targets inhibited by the Nepenthes extracts ( Table 2). The stem extract of N. miranda exhibited strong anti-KpSSB activity with an IC 50 value of 15.0 ± 1.8 µg/mL, while extracts of S. bullata did not cause a significant effect on the activity of KpSSB. SSB is required for DNA replication, and, thus, the pharmacological inhibition of bacterial SSB may be used to target pathogens. Indeed, suppression of DNA replication and metabolism is widely used as an antimicrobial strategy for antibiotic design. For example, quinolone and aminocoumarin antibiotics, which can be used to target bacterial DNA gyrase and topoisomerase IV, were successfully developed for clinical applications [47,48]. Thus, it is worth continuing the search for inhibitors against SSB.
Many valuable metabolites have been identified in different Nepenthes species [19]. It is assumed that the majority of the biological activities of Nepenthes species are related to naphthoquinones. For example, plumbagin derived from N. alata showed significant anticancer activity [20]. In animal models, plumbagin-treated mice showed a significant reduction in tumor growth, and no side effects [49]. In this study, the most abundant compound in the stem extract of N. miranda analyzed via GC-MS was plumbagin (12.9%), and, thus, the cytotoxic properties of this extract against Ca9-22 cells may be related to plumbagin. In addition, sitosterol, a phytosterol possessing significant anticancer activities against a variety of cancers [50,51], was the second most abundant compound (6.5%) found in the stem extract of N. miranda. Accordingly, the stem extract of N. miranda might induce apoptosis and cause cancer cell death mainly via the co-activities of plumbagin and sitosterol. The stem extract of N. miranda used was much more efficient against OSCC cells, i.e., Ca9-22 and CAL27 cells, than against PC9, 4T1, and B16F10 cells. Thus, it is of considerable interest to further explain why the cytotoxic specificity of this extract differs for possible medical applications. Given that OSCC is the most common subsite of head and neck cancer with a 5-year survival rate of only 50% [23], it is worth determining whether N. miranda extracts could be a potential natural alternative medication or complementary therapy, e.g., though its use as an adjuvant or ointment for the treatment of oral cancer.
Like the stem extract of N. miranda, the leaf extract of S. bullata also exhibited cytotoxicity in regard to cancer cell survival, migration, and proliferation, and induced apoptosis in cancer cells [37]. However, their contents, as tentatively identified via GC-MS, were found to be significantly different. Thus, the cytotoxic mechanisms may differ between these two extracts. Plumbagin, a major active compound in the stem extract of N. miranda, was not found in the leaf extract of S. bullata [37], but other naphthoquinones such as 7,8-dimethoxydunnione [52] and aggregatins [53] were found in the tuber extracts of similar Sinningia species. These and other naphthoquinones could possibly be useful in combination for further anticancer drug development.
The development of clinically useful small-molecule antibiotics has been a seminal event in the field of infectious diseases [10,12]. In this study, we found that the stem extract of N. miranda can strongly inhibit the ssDNA-binding activity of KpSSB (Figure 4). The 10 most abundant compounds in this extract, which are possibly responsible for the inhibition of KpSSB, were used for docking analysis (Figure 10). The compounds in the stem extract of N. miranda could occupy the ssDNA-binding sites of KpSSB; therefore, the KpSSB inhibitory capacity might derive from the co-effects of some of these compounds. SSB is essential for all aspects of DNA metabolism, such as DNA replication, repair, recombination, and replication restart [1,6,54], and thus potentially represents an attractive target in antipathogen chemotherapy. Previously, we found that the flavonol myricetin can bind to and inhibit PaSSB [27,29]. The binding site of the inhibitor myricetin was found to overlap with the ssDNA-binding sites in PaSSB [30,31]. Given that the crystal structure of KpSSB is similar to that of PaSSB [32,33], one might conclude that the PaSSB inhibitor myricetin must inhibit KpSSB. However, myricetin, as well as quercetin, kaempferol, and galangin, did not inhibit the ssDNA-binding activity of KpSSB ( Figure 3). Both residues K7 and E80, crucial myricetin and ssDNA-binding sites in PaSSB ( Figure 11A), are conserved in KpSSB (K8 and E81). However, other myricetin-interacting residues in PaSSB, namely I105, N106, G107, and N108, are not conserved in KpSSB ( Figure 11B,C). Correspondingly, these residues in KpSSB are V106, G107, G108, and T109 ( Figure 11A). This may explain why myricetin cannot inhibit KpSSB because the myricetin-binding residues are significantly different in KpSSB. For example, superposition analysis ( Figure 11D) indicated that the myricetin-interacting distances of residues G107 and T109 in KpSSB (subunit C) were 6.0 and 5.5 Å, respectively ( Figure 11E). Based on these distances (>5 Å), these two residues in KpSSB may play no role in myricetin binding. However, this speculation must be further biochemically and structurally investigated.
liferation, and migration of Ca9-22 cells were also examined. The most abundant compounds in this extract were determined via GC-MS to obtain a better understanding of which compounds may be active, alone, or acting in combination in these biological activities. These results may indicate the pharmacological potential of the stem extract of N. miranda for possible medical applications.  In conclusion, this study was the first to identify the anti-KpSSB capacity of the stem extract of N. miranda. The cytotoxic effects of this extract on the survival, apoptosis, proliferation, and migration of Ca9-22 cells were also examined. The most abundant compounds in this extract were determined via GC-MS to obtain a better understanding of which compounds may be active, alone, or acting in combination in these biological activities. These results may indicate the pharmacological potential of the stem extract of N. miranda for possible medical applications.

Recombinant Protein Expression and Purification
Briefly, the KpSSB expression plasmid pET21b-KpSSB [57][58][59] was transformed into E. coli BL21 (DE3) cells. The resultant cells were grown to OD 600 of 0.9 in LB medium containing 250 µg/mL ampicillin with rapid shaking at 37 • C. The overexpression was induced by incubating with 1 mM isopropyl thiogalactopyranoside for 9 h at 25 • C. The cells overexpressing the protein were chilled on ice, harvested by centrifugation, resuspended in Buffer A (20 mM Tris-HCl, 5 mM imidazole and 0.5 M NaCl, pH 7.9) and disrupted by sonication with ice cooling. The recombinant protein was purified from the soluble supernatant using Ni 2+ -affinity chromatography (HisTrap HP; GE Healthcare Bio-Sciences, Piscataway, NJ, USA), eluted with Buffer B (20 mM Tris-HCl, 250 mM imidazole, and 0.5 M NaCl, pH 7.9), and dialyzed against a dialysis buffer (20 mM HEPES and 100 mM NaCl, pH 7.0; Buffer C). Protein purity remained at >97% as determined by SDS-PAGE (Mini-PROTEAN Tetra System; Bio-Rad, Hercules, CA, USA).

EMSA
dT20, dT30, and dT35 were biotinylated at the 5 terminal and incubated with purified KpSSB of different concentrations (0, 20, 39, 78, 156, 312, 625, 1250, 2500, and 5000 nM). Including dsDNA substrates PS4/PS3, PS4/PS3-3 -dT25, and PS4/PS3-3 -dT30, the final concentration of these DNA substrates for analysis was 30 fmol/µL. EMSA was performed using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Waltham, MA, USA) [27,28]. In brief, KpSSB was incubated for 60 min at 37 • C with DNA substrate in a total volume of 6 µL in 40 mM Tris-HCl (pH 7.5) and 50 mM NaCl. Following incubation, 4 µL of a dye mixture (0.01% bromophenol blue and 40% glycerol) was added. The resulting samples were loaded and resolved on native polyacrylamide gel (8%) at 100 V for 1 h in TBE running buffer (89 mM Tris borate and 1 mM EDTA). The protein-DNA complexes were electroblotted to positively charged nylon membrane (GE, Boston, MA, USA) at 100 V for 30 min in TBE buffer. Transferred DNA was cross-linked with nylon membrane using a UV-light cross-linker instrument equipped with 312 nm bulbs for a 10 min exposure. Using streptavidin-horseradish peroxidase conjugate and chemiluminescent substrate contained in SuperSignal™ West Atto Ultimate Sensitivity Substrate (Pierce Biotechnology, Waltham, MA, USA), biotin-labeled DNA was detected. The DNA-binding ability of KpSSB ([Protein] 50 ) was estimated through linear interpolation from the concentration of the protein that bound 50% of the input DNA.

Inhibition Assay
EMSA for an inhibition test against KpSSB was conducted using the protocol described previously [27,28]. A biotinylated dT30 was used as substrate for this inhibition assay. KpSSB (625 nM) was incubated with dT30 and the indicated compound (0, 37.5, 100, and 300 µM) for 60 min at 37 • C. Indicated plant extracts (7.8-1000 µg/mL) were also included in the binding assay for an inhibition test against KpSSB. Following incubation, the resultant KpSSB solution was analyzed using EMSA. The titration curves were generated and the concentration of the compound or plant extract required for 50% inhibition (IC 50 ) was determined directly from the graphic analysis.

Plant Materials and Extract Preparations
Briefly, N. miranda (leaf, stem, and pitcher) [16] and S. bullata (leaf, stem, and tuber) [37], obtained at Guoguang Flower Market and Taiwan Provincial Flower Marketing Cooperative and identified by Dr. Zhong-Bao Zhang in December 2020, were collected, dried, cut into small pieces, and pulverized into powder. Extractions were carried out by placing 1 g of plant powder into 250 mL conical flask. A total of 100 mL of methanol was added to the flask and shaken on an orbital shaker for 5 h. The resultant extract was filtered using a 0.45 µm filter. Solvent (methanol) in extracts was removed via a hot air circulation oven at 40 • C. Extracts were stored at −80 • C until use. The extract powder was dissolved in 20% DMSO to make a stock solution at a concentration of 20 mg/mL. For the anticancer cell assays, the stock was diluted with the supplemented culture medium to the indicated assay concentrations. The cancer cells were incubated with the resultant extract solutions or the culture medium with 0.2% DMSO, reference as treatment, or the control group. For EMSA, the stock was diluted with Buffer B to the indicated assay concentrations. The protein was incubated with the resultant extract solutions or Buffer B with 1% DMSO, reference as treatment, or the control group.

GC-MS Analysis
The molecular composition of samples was determined using GC-MS analysis. The Thermo Scientific TRACE 1300 Gas Chromatograph with a Thermo Scientific ISQ Single Quadrupole Mass Spectrometer System was used to analyze the filtered sample. A Rxi-5ms column (30 m × 0.25 mm i.d. × 0.25 µm film) was used. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The initial oven temperature was 40 • C. It was maintained at 40 • C for 3 min, and then the temperature was gradually increased to 300 • C at a rate of 10 • C/min. This temperature was maintained for 1 min. The temperature of the injection port was 300 • C and the flow rate of helium was 1 mL/min. The compounds discharged from the column were detected using a quadrupole mass detector. The ions were generated by electron ionization method. The temperatures of the MS quadrupole and source were 150 and 300 • C, respectively, the electron energy was 70 eV, the temperature of the detector was 300 • C, the emission current multiplier voltage was 1624 V, the interface temperature was 300 • C, and the mass range was from 29 to 650 amu. The peak area normalization method was used to determine the relative mass fraction of each chemical component. Compounds were identified by matching generated spectra with NIST 2011 and Wiley 10th edition mass spectral libraries.

Trypan Blue Cytotoxicity Assay
The trypan blue cytotoxicity assay was performed to assess cell death [38]. Briefly, Ca9-22, CAL27, B16F10, PC-9, and 4T1 cells (1 × 10 4 ) were incubated with the extract of N. miranda in a 100 µL volume. After 24 h, a trypan blue staining assay was used to estimate the cytotoxic activity exhibited by the extract.

Chromatin Condensation Assay
The apoptosis in cancer cells was assayed with Hoechst 33342 staining [39]. Briefly, Ca9-22, CAL27, B16F10, PC-9, and 4T1 cells were seeded in 96-well plates at a density of 5 × 10 3 cells per well. Culture medium was 200 µL per well. Cells were allowed to adhere for 16 h. Cells were incubated with the extract of N. miranda for 24 h, washed with PBS, and stained with the Hoechst dye (1 µg/mL) in the dark at RT for 10 min. ImageXpress Pico (Molecular Devices, Silicon Valley, CA, USA) was used to image cells. Image acquisition was performed on each well using a 20× magnification, 6 × 6 square image scan, on the DAPI filter cubes. Image analyses were performed using the CellReporterXpress Version 2 software. The apoptotic index was calculated as follows: apoptotic index = apoptotic cell number/(apoptotic cell number + nonapoptotic cell number).

Clonogenic Formation Assay
To assess the inhibition of Ca9-22 cell proliferation, a clonogenic formation assay [61] was used. Briefly, Ca9-22 cells were seeded at a density of 1 × 10 3 cells per well into 6-well plates and incubated overnight for attachment. The cells were incubated with the extract of N. miranda for 5-7 days. After washing with PBS, formed colonies were fixed with methanol and stained with 0.5% crystal violet for 20 min. The number of colonies was counted under a light microscope.

Wound-Healing Assay
To analyze the inhibition of Ca9-22 cell migration, the wound-healing assay [62] was performed. Briefly, Ca9-22 cells were seeded in 96-well plates and incubated in serumreduced medium for 6 h. The cells were then wounded in a line across the well with a 200 µL pipette tip and washed twice with the serum-reduced medium. The cells were treated with the extract of N. miranda and incubated for 24 h to allow migration.

Flow Analysis
Flow cytometry was used for cell cycle analysis. Ca9-22 cells were treated with DMSO or the extract of N. miranda for 24 h and harvested with trypsin. The harvested cells were washed, resuspended in PBS with 1% FBS, and then fixed with cold ethanol (70%). Fixed cells were washed and incubated in PBS buffer for 5 min. The resultant cells were resuspended in PI/RNase solution (PBS, RNase, and 50 µg/mL PI) and stained for 30 min at 37 • C in the dark. A BD FACSCanto II (BD Biosciences, San Jose, CA, USA) was used to analyze these treated cells. Through FlowJo v10 software (Tree Star, Inc., Ashland, OR, USA), the distribution of each phase was calculated and visualized.