Monascus Pigment Rubropunctatin: A Potential Dual Agent for Cancer

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Monascus pigment rubropunctatin: a potential dual agent for cancer chemotherapy and phototherapy Yunquan Zheng, Yun Zhang, Deshan Chen, Haijun Chen, Ling Lin, Chengzhuo Zheng, and Yanghao Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05343 • Publication Date (Web): 08 Mar 2016 Downloaded from http://pubs.acs.org on March 10, 2016

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Journal of Agricultural and Food Chemistry

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Monascus pigment rubropunctatin: a potential dual agent for cancer

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chemotherapy and phototherapy

3

Yunquan Zheng,*,†,§ Yun Zhang,†,§ Deshan Chen,† Haijun Chen,*,† Ling Lin,†

4

Chengzhuo Zheng,† Yanghao Guo§

5

6

†

College of Chemistry, Fuzhou University, 2 Xueyuan Road, Fuzhou 350116, China

7

§

Fujian Key Laboratory of Medical Instrument and Pharmaceutical Technology,

8

Fuzhou University, 523 Gongye Road, Fuzhou 350002, China

9

Corresponding authors:

10

* Yunquan Zheng, PhD

11

College of Chemistry

12

Fuzhou University

13

Fuzhou, Fujian 350116, China

14

Email: [email protected]

15

*Haijun Chen, PhD

16

College of Chemistry

17

Fuzhou University

18

Fuzhou, Fujian 350116, China

19

Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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The Monascus pigment, rubropunctatin, was extracted and purified from red mold

22

rice (RMR) and its cytotoxic activities against human cervical carcinoma HeLa cells

23

were studied under the conditions with or without light irradiation. The IC50 value of

24

rubropunctatin against HeLa cells in the dark was 93.71 ± 1.96 µM (24 h), while the

25

cytotoxic activity was enhanced more than 3 times (IC50 = 24.02 ± 2.17 µM) under

26

light irradiation(halogen lamp: 500 W, wavelength: 597-622 nm, fluence rate: 15 mW

27

cm-2, for 30 min). However the IC50 value of rubropunctatin against the immortalized

28

human cervical epithelial H8 cells was more than 300 µM even under light irradiation,

29

indicating that rubropunctatin has a favorable selectivity index (SI). Treatment of

30

HeLa cells with rubropunctatin in the dark or under light irradiation resulted in a dose

31

dependent apoptosis, as validated by the increase in the percentage of cells in sub-G1

32

phase and phosphatidylserine externalization, and the inductive effect on HeLa cell

33

apoptosis was boosted by the light irradiation. In addition, treatment with

34

rubropunctatin alone or under light irradiation was found to induce apoptosis in HeLa

35

cells via the mitochondrial pathway, including loss of mitochondrial membrane

36

potential, activation of caspase-3, -8 and -9, and increase of the level of intracellular

37

ROS. It was suggested that rubropunctatin could be a promising natural dual

38

anticancer agent for photodynamic therapy and chemotherapy.

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KEYWORDS: Monascus, rubropunctatin, chemotherapy, phototherapy

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INTRODUCTION

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Cancer has become a significant public health problem due to its high rates of

43

morbidity and mortality. Recently, development of innovative anticancer drugs with

44

enhanced efficacy and improved selectivity is one of the most important works in

45

medicinal chemistry research communities. This paradigm has resulted in the

46

successful development of numerous anticancer agents. However, almost all kinds of

47

the existing chemotherapeutic drugs in the clinical application for cancer treatment

48

often result in therapeutic resistance and undesired side effects in all types of cancers.

49

In addition, it is reported that cancer therapy relying on a single therapeutic treatment

50

is not always reliable due to the complex network of cellular pathways.1 Therefore, it

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is still a big challenge to find a suitable therapy for human cancer treatment.

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To date, combination therapy has been considered as a promising strategy to take

53

the advantage of each treatment to minimize undesirable adverse effects and improve

54

therapeutic efficiency.2,

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phototherapy has emerged as an effective strategy for cancer therapy.4-6 Phototherapy

56

also known as photodynamic therapy (PDT) is a well-established clinical cancer

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treatment that involves a certain photosensitizer, light and molecular oxygen.7, 8 Upon

58

irradiation with the light of an appropriate wave-length, the excited photosensitizer

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transfers energy from the ground state to the excited state to generate highly reactive

60

species, leading to irreversible damage of cancer cells. Recently, several

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nanoparticle-based delivery systems including silica nanocages and lipid-polymers

3

In particular, the combination of chemotherapy and



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have been developed to release the chemotherapeutic drug and photosensitizer

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simultaneously in the tumour region to exert the synergistic anticancer effect.9-11

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Although these important progresses have demonstrated the obvious potential both in

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vitro and in vivo, there have been few report of the small organic molecule acting as

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both chemotherapeutic agent and photosensitizer.7, 8, 12

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Monascus is a versatile genus that can be used for the production of various

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metabolites and is useful as food additives and pharmaceuticals. 13, 14 In our previous

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work six pigment components were successfully separated from Monascus product,

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which were two yellow pigments (monascin and ankaflavin), two orange pigments

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(rubropunctatin and monascorubrin) and two red pigments (rubropunctatamine and

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monascorubramine). The cytotoxicity of the Monascus pigments to various human

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cancer cells (SH-SY5Y, HepG2, HT-29, BGC-823, AGS, and MKN45) was separately

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evaluated. rubropunctatin showed the highest anticancer effect within the tested

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compounds. The inhibition effect of rubropunctatin was higher than that of taxol on

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the growth of the human gastric cancer cell SH-SY5Y (P < 0.05), BGC-823 (P yellow light > green light > blue light (Table 1 ).

247

Cytotoxicity of Rubropunctatin to HeLa Cells under Conditions with or without

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Irradiation. To validate whether rubropunctatin could be used as a photosensitizer

249

against cancer cells, we investigated the cytotoxic effect of rubropunctatin on the

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growth of HeLa cells under conditions with or without light irradiation.

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rubropunctatin showed an obvious concentration-dependent inhibition effect on HeLa

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cells from 3 to 120 µM under conditions either with or without light


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irradiation. rubropunctatin inhibited the proliferation of HeLa cells in the dark with an

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IC50 of 93.71 ± 1.96 µM after 24 h incubation. The experimental data indicated that

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rubropunctatin alone has a modest inhibitory effect on HeLa cells. In the presence of

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light irradiation, rubropunctatin displayed a remarkable growth inhibitory effect

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against HeLa cells with an IC50 of 24.02 ± 2.17 µM after 24 h incubation. It was

258

suggested that rubropunctatin could be used as a promising natural dual anticancer

259

agent for photodynamic therapy and chemotherapy.

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A time-course study revealed that rubropunctatin in the dark displayed no toxic

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effect on HeLa cells at the first 4 hours however it obviously inhibited the

262

proliferation of HeLa cells after 12 h or 24 h incubation (Table 2). Rubropunctatin

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decreased the viability of HeLa cells in a time-dependent manner under conditions

264

with or without light irradiation.

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As shown in Table 2, Taxol was used as a control and its cytotoxicity against Hela

266

cells was determined. Under conditions without irradiation, Taxol showed a good

267

inhibition effect on the growth of Hela cells with IC50 of 73.31±2.23µmol/L at 12h

268

and 44.32±3.84µmol/L at 24h. However, no obvious change was observed, compared

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with the determined data in the absence of light irradiation with those in the presence

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of light irradiation, IC50 of 76.86±2.54µmol/L at 12h and 42.71±4.31µmol/L at 24h.

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It was notable that under conditions with light irradiation, the inhibition effect on

272

the treated Hela cells with rubropunctatin was higher than that with taxol. In addition,

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rubropunctatin was basically no-cytotoxic to the immortalized human cervical



274

epithelial H8 cells with the IC50 value greater than 300 µM (at 24h) in the dark or

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under light irradiation, indicating that rubropunctatin has a favorable selectivity index

276

(SI).

277

Apoptosis of HeLa Cells Induced by Rubropunctatin under Conditions with or

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without Light Irradiation. After being treated separately with different

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concentrations of rubropunctatin for 24 h, the AO/EB staining of HeLa cells was

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performed to evaluate the mode of cell death under the conditions with or without

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light irradiation. The cell morphology was observed under the fluorescence

282

microscope. Figure 3 showed clear morphological changes in the nucleolus, internal

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organelles and plasma membrane integrity caused by rubropunctatin in a

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concentration-dependent manner. In the presence or absence of light irradiation, the

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rubropunctatin untreated HeLa cells showed uniform green fluorescence with normal

286

morphology, indicating that light irradiation alone did not change morphology of the

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cells. After rubropunctatin treatment without or with light irradiation, extensive

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nuclear margination accompanied by chromatin condensation and fragmentation,

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indicative of apoptotic cell death, was observed in the treated cells.23, 24 With the

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increase of rubropunctatin concentration, the marked nuclear condensation, membrane

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breakage, nuclear fragmentation and apoptotic bodies became visibly dominant and

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fluorescence turned into orange, indicating cell death.

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HeLa cells which were incubated with 30 µM rubropunctatin in the absence of light

294

irradiation showed green fluorescence. It demonstrated that the cell membranes were


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normally integrated and kept EB out of the cells. When treated with the same

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concentration of rubropunctatin in the presence of light irradiation, the fluorescence

297

turned into orange and showed that the membranes were impaired. The results of

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AO/EB nuclear staining indicated remarkable induction of apoptosis in HeLa cells by

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rubropunctatin under the conditions with light irradiation.

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Cell Cycle Analysis. To characterize cell death induced by rubropunctatin, cell cycle

301

analysis was performed by staining the cells with PI (Table 3). HeLa cells were

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treated separately with different concentrations of rubropunctatin (0, 3, 30, 60, 90 and

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120 µM) under the conditions with or without light irradiation. The data showed that

304

treatment with rubropunctatin increased the fraction of cells in the sub-G1 phase (an

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apoptotic phenomenon) in a dose-dependent manner under light irradiation, while

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treatment with rubropunctatin in the dark only enhanced the fraction of cells in the

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sub-G1 phase at the high concentration. However, no arrest at any phase of the cell

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cycle was found. It was hypothesized that rubropunctatin might inhibit cell growth of

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the HeLa cells through induction of apoptosis, and the affection was enhanced by

310

light irradiation.

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Apoptotic Analysis of HeLa Cells treated with Rubropunctatin under the

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Conditions with or without Light Irradiation. The cytotoxic effect of chemo-

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photodynamic treatment on HeLa cells was further quantified by flow cytometry.

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HeLa cells were double-labelled by Annexin-V/PI after they were treated with

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different concentrations of rubropunctatin under the conditions with or without light



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irradiation. The Annexin-V and PI positive cells were defined as late

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apoptotic/necrotic stage. As shown in Figure 4, the untreated cells were primarily

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Annexin-V and PI negative, indicating that they were viable and not undergoing

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apoptosis either in the absence or presence of light irradiation. After treatment with

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rubropunctatin in the dark or under light irradiation, rubropunctatin produced a

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dose-dependent increase in the HeLa necrotic population and a decrease in viable

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population, and induced HeLa cells from early apoptotic stage into late

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apoptotic/necrotic stage. Compared with the treatment in the dark, the results showed

324

that rubropunctatin plus light marginally induces cell late apoptosis/necrosis (such as

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38.6% vs 30.2% at 120 µM), indicating that the cytotoxic effect of rubropunctatin on

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HeLa cells was boosted by the light irradiation.

327

Rubropunctatin-induced Loss of ∆Ψm in HeLa Cells under the Conditions with

328

or without Light Irradiation. The loss of mitochondrial membrane potential (∆Ψm)

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is regarded as a limiting factor in the induction of apoptosis by the intrinsic pathway.25

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To determine whether rubropunctatin induced damage of the mitochondrial function

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with light irradiation, we determined ∆Ψm by using JC-1 staining. The absolute

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red/green JC-1 intensity ratio (FL1/FL2) was measured.

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quantitative analysis of mitochondrial membrane depolarization by flow cytometry

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showed that the cellular ∆Ψm was decreased after exposure of HeLa cells to

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rubropunctatin under the conditions with or without light irradiation. The negative

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control HeLa cells were with higher potentials and theratio of red/green JC-1

337

fluorescence was above 1.3. In the positive control, CCCP was used as an inducer to


As shown in Table 4,

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decrease the mitochondrial membrane potential and the determined FL1/FL2 was 0.87.

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After HeLa cells were exposed to 30 or 60 µM rubropunctatin for 24 h, it was noted

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that ∆Ψm did not change ether in the dark or under light irradiation, indicating that the

341

loss of ∆Ψm possibly reached a maximum at 30 µM. These results indicated that

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rubropunctatin induced apoptosis via the mitochondrial pathway. The cytotoxic effect

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of rubropunctatin on HeLa cells was boosted by the light irradiation, but

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mitochondrial membrane potential did not change.

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Induction of the Activation of Caspase-3, -8 and -9 by Rubropunctatin. Treatment

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of HeLa cells with rubropunctatin (30 and 60 µM) resulted in a dose-dependent

347

increase in the activities of caspase-3, -8 and -9 either in the dark or under light

348

irradiation (Figure 5). Compared with the treatment in the dark, the data under the

349

conditions with light irradiation showed an obvious increase of the activity of

350

caspase-3, -8 and -9, further indicating that the combination of rubropunctatin

351

treatment and light irradiation was more effective than treatment alone with the drug

352

to induce cell apoptosis.

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The mitochondrial pathway plays an essential role for cell apoptosis, in which

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caspase-3, -8 and -9 are involved. Our experimental data demonstrated that

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rubropunctatin treatment led to the activation of caspase-3, -8 and -9. These results

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indicated that rubropunctatin induced cell apoptosis via the mitochondrial pathway.

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Production of Cellular ROS induced by Rubropunctatin under Conditions with

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or without Light Irradiation. The interaction of the photosensitisers with cancer



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cells can induce oxidative stress by enhancing the production of intracellular ROS

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over the cellular antioxidant defenses. To investigate the effect of rubropunctatin on

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the production of intracellular ROS, the treated cells were quantified by determining

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the percentage of cells with increased green fluorescence in a flow cytometer. Under

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the conditions without light irradiation the determined ROS levels produced in the

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Hela cells treated by rubropunctatin with lower concentrations were in the same order

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of magnitude with the blank group of rubropunctatin untreated cells. It is evident from

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the flow cytometric analysis that the treatment of rubropunctatin raised the level of

367

intracellular ROS only at the high concentration of 90µM in the absence of irradiation.

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However, the intracellular ROS level in presence of light irradiation increased even at

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the lower rubropunctatin concentrations, which was toxic enough to augment the

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apoptotic cell death by damaging mitochondrial membrane integrity. Table 5 showed

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an obvious elevation of the ROS production in the rubropunctatin treated cells under

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the conditions with light irradiation compared to without irradiation. The experimental

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data of the intracellular ROS level treated by rubropunctatin under conditions with

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light irradiation or without light irradiation were consistent with the reported data

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regarding the cytotoxic activities against HeLa cells mentioned above.

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In this work, we found the photochemical properties of rubropunctatin and reported

377

the fact that light irradiation with wavelength 597-622nm remarkably strengthened its

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cytotoxic activities against human cervical carcinoma HeLa cells. The cytotoxicity of

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rubropunctatin on various human cervical carcinoma cells (Siha, Caski, C33A) was

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also systematically evaluated in our experiments, and the tendency of the cytotoxicity


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under the conditions with irradiation over that without irradiation was in the same

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manner as Hela cells. Photodynamic therapy is believed to cause cell damage via the

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production of ROS and subsequently to induce apoptotic signaling via the

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mitochondrial pathway. Rubropunctatin increased intracellular ROS generation and

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decreased mitochondrial membrane potential. These results suggest that the anticancer

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effect of rubropunctatin was probably due to the modulation of cell signaling and

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intracellular ROS generation. The treatment of rubropunctatin under the conditions

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with light irradiation promoted the induction of Hela cells into late apoptosis/necrosis

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phase. Activation of caspase 8 and 9 in the upstream resulted in the activation of

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caspase 3 in the downstream and in the end speeded up cell apoptosis. In order to

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develop anticancer effects of rubropunctatin, it is essential to further understand the

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precise mechanism of cytotoxic activities of rubropunctatin in the presence of light

393

irradiation.

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The photochemical reaction of rubropunctain was studied by HPLC-MS (data not

395

shown).

Rubropunctain

is a

natural azaphilone compound

396

microbiological metabolite, which has a typical structure with multiple rings

397

involving multiple conjugated double bonds and mixed oxygen atoms. After light

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irradiation, part of rubropunctain was degradated and some new compounds with

399

higher polarity were produced. The hydroxyl free radicals and hydrogen free radiacals

400

produced from RH (hydrogen donor) under the conditions with light irradiation could

401

easily attack the conjugating double bands in the rubropunctain molaculars. When

402

rubropunctain in the water solution was radiated with light, colour fading was


isolated

from


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observed, which was due to the conjugate structure’s damage. It is required to further

404

explore the photochemical reaction mechanism of rubropunctain in the presence of

405

light irradiation.

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AUTHOR INFORMATION

407

Corresponding Authors

408

*Fax:

409

[email protected] (Y. Zheng)

410

Fax: +86-591-22866234 ; Tel: +86-591-22866227; E-mail: [email protected] (H.

411

Chen)

412

Notes

413

The authors declare no competing financial interest.

414

Acknowledgment

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This work was supported by National Natural Science Foundation of China (No.

416

J1103303),

417

2012J05155), Marine Public Welfare Research Project of China (201205022), and the

418

Technology Development Foundation of Fuzhou University (Project Numbers

419

2011-XY-7 and 2013-XQ-9).

420

REFERENCES

421

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+86-591-83720772;

Tel:

Natural Science Foundation

+86-591-83720772;

of Fujian Province

of

E-mail:

China

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Lovell, J. F.; Liu, T. W.; Chen, J.; Zheng, G., Activatable photosensitizers for imaging and therapy.

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nanocages for cancer cell detection and the combined chemotherapy and photodynamic therapy. ACS applied materials & interfaces 2011, 3, 2479-86. 10. Zheng, M.; Yue, C.; Ma, Y.; Gong, P.; Zhao, P.; Zheng, C.; Sheng, Z.; Zhang, P.; Wang, Z.; Cai, L., Single-step assembly of DOX/ICG loaded lipid--polymer nanoparticles for highly effective chemo-photothermal combination therapy. ACS nano 2013, 7, 2056-67. 11. Ren, H.; Wu, Y.; Li, Y.; Cao, W.; Sun, Z.; Xu, H.; Zhang, X., Visible-Light-Induced Disruption of Diselenide-Containing

Layer-by-Layer

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Photodynamic Therapy. Small 2013, 9, 3981-6. 12. Chen, H.; Zhou, X.; Gao, Y.; Zheng, B.; Tang, F.; Huang, J., Recent progress in development of new sonosensitizers for sonodynamic cancer therapy. Drug discovery today 2014, 19, 502-9. 13. Osmanova, N.; Schultze, W.; Ayoub, N., Azaphilones: a class of fungal metabolites with diverse biological activities. Phytochem Rev 2010, 9, 315-342 14. Gao, J. M.; Yang, S. X.; Qin, J. C., Azaphilones: chemistry and biology. Chemical reviews 2013, 113, 4755-811. 15. Zheng, Y.; Xin, Y.; Shi, X.; Guo, Y., Cytotoxicity of Monascus pigments and their derivatives to human cancer cells. Journal of agricultural and food chemistry 2010, 58, 9523-8. 16. Zheng, Y.; Xin, Y.; Shi, X.; Guo, Y., Anti-cancer effect of rubropunctatin against human gastric carcinoma cells BGC-823. Applied microbiology and biotechnology 2010, 88, 1169-77. 17. Wild, D.; Toth, G.; Humpf, H. U., New monascus metabolites with a pyridine structure in red fermented rice. Journal of agricultural and food chemistry 2003, 51, 5493-6. 18. Su, N. W.; Lin, Y. L.; Lee, M. H.; Ho, C. Y., Ankaflavin from Monascus-fermented red rice exhibits selective cytotoxic effect and induces cell death on Hep G2 cells. Journal of agricultural and food chemistry 2005, 53, 1949-54. 19. Hong, M. Y.; Seeram, N. P.; Zhang, Y.; Heber, D., Anticancer effects of Chinese red yeast rice versus monacolin K alone on colon cancer cells. The Journal of nutritional biochemistry 2008, 19, 448-58. 20. Ho, B. Y.; Pan, T. M., The Monascus metabolite monacolin K reduces tumor progression and



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480

481


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482 483

Table 1. The influence of light irradiation with different wavelengths on the light chemical reaction of rubropunctatin Irradiation condition

wavelength

Degradation Rate of rubropunctain, %

red light

622-770nm

64.5

orange light

597-622nm

68.8

yellow light

577-597nm

58.4

green light

492-577nm

57.2

blue light

455-492nm

16.3

484



485

Page 24 of 33

Table 2. Cytotoxicity of rubropunctatin and Taxol on HeLa and H8 cells

Cells treated with rubropunctatin for different time (h)

IC50 of rubropunctatin (µmol/L)

IC50 of Taxol (µmol/L)

Without Light Irradiation

With Light Irradiation

Without Light Irradiation

With Light Irradiation

HeLa (4h)

>1000

>1000

>1000

>1000

HeLa (12h)

146.61±2.45

44.19±2.86

73.31±2.23

76.86±2.54

HeLa (24h)

93.71±1.96

24.02±2.17

44.32±3.84

42.71±4.31

H8 (24h)

>300

>300

120.83±4.52

124.39±5.03

486


Page 25 of 33


Table 3. DNA flow cytometric analysis of cell cycle distribution.a

487

Condition without light irradiation with light irradiation

Cell

Concentration (µM) Control

3

30

60

90

Sub-G1

1.64±0.02

1.98±0.04*

1.91±0.01*

1.96±0.03*

2.6±0.03**

6.4±0.01**

G0/G1

63.95±5.21

63.83±3.43

65.82±5.42

61.26±6.54

59.1±7.54*

60.95±3.54*

S

9.94±0.89

9.22±0.45

9.88±0.52

8.92±0.23*

9.30±0.53

10.17±0.48

Cycle

120

G2/M

24.47±1.23

24.97±1.14

22.39±1.42

27.86±0.95*

29.00±0.92*

22.48±1.42*

Sub-G1

2.11±0.03

3.25±0.01**

5.03±0.03**

8.21±0.06**

12.2±1.03**

14.09±1.40**

G0/G1

63.79±5.06

63.43±3.40

57.98±5.03*

56.2±5.04**

52.1±4.05**

47.36±6.95**

S

10.66±1.03

10.67±1.34

11.33±2.01

10.91±1.24

10.23±0.89

9.3±0.98*

G2/M

23.44±1.04

22.65±1.93

25.66±1.02*

24.68±1.29

25.47±2.01*

29.25±2.89**

488

a

489

the respective controls for each experimental test condition was assessed using Student’s unpaired

490

t test, with *p < 0.05 or **p < 0.01 being regarded as statistically significant.

Each value represents the mean ± SD of three separate experiments. A significant difference from

491



492

Table 4. Effects of rubropunctatin on mitochondrial membrane potential (∆Ψm) of

493

Hela cells without or with light irradiationa. Condition

Concentration(µM) b

without light irradiation with light irradiation

CCCP control 30 60 CCCPb control 30 60

FL2/FL1 0.87±0.09** 1.37±0.12 0.86±0.05** 0.87±0.04** 0.78±0.02** 1.41±0.11 0.94±0.06** 0.94±0.05**

494

a

495

the respective controls for each experimental test condition was assessed using Student’s unpaired

496

t test, with *p

Monascus Pigment Rubropunctatin: A Potential Dual Agent for Cancer

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