Inhibitory Effect of Phycocyanin Combined with Photodynamic Therapy in Sw1990 Pancreatic Cancer Cells

 Abstract: [Objective] To investigate the inhibitory effect of C-PC combined with photodynamic therapy on SW1990 pancreatic cancer cells, and to explore the mechanism of its action in terms of immunomodulation. ［Methods] The experiment was divided into 6 groups: normal group, model group, CY group, laser irradiation group, phycocyanin group (C-PC group) and phycocyanin+laser irradiation group (C-PC+laser irradiation group). Except for the normal group, the remaining groups were inoculated with SW1990 cells in the axilla of the right forelimb of mice to construct mouse xenograft tumor models. After successful modeling, the tumor diameter was measured, the spleen weight was weighed, and the splenic index, tumor volume and tumor inhibition rate were calculated. The levels of TNF-α and IL-6 in the serum and culture fluid of mouse splenocytes were determined by ELISA. ［Compared with the model group, the tumor weight of the C-PC group was significantly reduced (P<0.05); the tumor weight and tumor volume of the CY group and the C-PC+laser irradiation group were significantly reduced (P<0.01 and P<0.05, respectively); and the laser irradiation group did not have any significant effect on the tumor weight and tumor volume. The tumor inhibition rates of the laser irradiation group, C-PC group and C-PC+laser irradiation group were (39.42±8.47)%, (42.85±12.15)% and (74.80±11.85)%, respectively, and the tumor inhibition rate of the C-PC+laser irradiation group was comparable to that of the CY group [(78.58±8.87)%].

 

Compared with the normal group, the spleen weight and spleen index of the model group were significantly reduced (P<0.05); compared with the model group, the laser irradiation group, the C-PC group and the C-PC+laser irradiation group could significantly increase the spleen weight and spleen index (P<0.05 or P<0.01), while the spleen weight and spleen index of the CY group of mice were significantly decreased (P<0.05). Compared with the normal group, there was no significant difference in the levels of TNF-α and IL- 6 in the model group (P>0.05); compared with the model group, the C-PC group and the C-PC+laser irradiation group significantly increased the levels of TNF-α and IL-6 in the mice (P<0.05 or P<0.01); whereas, in the CY group, there was a significant decrease in the levels of TNF-α and IL-6 (P<0.05). P<0.05 or P<0.01); the laser irradiation group promoted IL-6 (P<0.05), but had no significant effect on TNF-α (P>0.05). [Conclusion] The combination of photodynamic therapy with phycocyanin can effectively inhibit tumor growth, improve the immune function of the spleen as an immune organ in mice with cancer, enhance the expression of TNF-α and IL-6 in the serum of mice with cancer as well as in the supernatant of splenocyte culture fluid, and enhance the body's humoral immunity, suggesting that the combination of photodynamic therapy with phycocyanin can achieve the purpose of inhibiting tumor growth by regulating the body's immune function. achieve the purpose of inhibiting tumor growth.

 

Spirulina platensis is a multicellular filamentous spiral-shaped marine alga rich in C-phycocyanin (C-PC), polysaccharides, β-carotene and other bioactive components. Among them, C-phycocyanin is a major functional protein in Spirulina, accounting for 20% of the dry basis of Spirulina [1], and it is a photosensitizer capable of producing living oxygen under light excitation for photodynamic therapy [2], which has the pharmacological activities of enhancing immunity, anti-tumor, anti-oxidation, scavenging of free radicals, anti-inflammation, and protection of liver, and it has the potential to be developed into a great number of medicines [3-5].

 

Photodynamic therapy (PDT) is one of the most effective treatments for tumors. PDT can lead to tumor cell death and apoptosis, as well as damage to tumor-associated blood vessels. In recent years, the mechanism of action of PDT has received increasing attention. The immune escape of tumor cells is an important factor in tumor development [6-7]. At present, many relevant studies have confirmed that phycocyanin combined with photodynamic therapy in the treatment of solid tumors has achieved excellent therapeutic effects [8-10]. In this study, we investigated the in vivo antitumor effects of phycocyanin combined with photodynamic therapy on human pancreatic cancer cells from the perspective of immunoregulation, which will provide a certain experimental basis for the development of antitumor drugs and the development and application of phycocyanin as a photosensitizer for the treatment of clinical cancer diseases.

 

1 Experimental materials

1.1 Animals   

 SPF grade BALBC/C nude mice, 40 mice, half male and half female, body mass 18-20 g, purchased from Hunan Slake Kingda Laboratory Animal Co. The animals were kept in a sterile environment.

 

1.2 Cell lines   

Human pancreatic cancer cells SW1990 were purchased from Kunming Institute of Zoology, Chinese Academy of Sciences.

 

1.3 Drugs and reagents   

Spirulina obtususifera algal blue protein lyophilized powder, purity of A620/A280 ≥ 3.5, purchased from Ningbo Binmei Biotechnology Company, sealed in a cool, dry and light-proof place for preservation, before use, with sterilized distilled water to the required concentration; cyclophosphamide for Injec- tion (cyclophosphamide for CY, Jiangsu Shengdi Pharmaceutical Co.    Ltd.) was purchased from Ruikang Hospital affiliated with Guangxi University of Traditional Chinese Medicine, 0.2 g per vial, stored at a temperature not exceeding 25 ℃, and prepared in sterilized saline to the required concentration before use; incomplete Leibovitz's L-15 medium (No.: KGM41300N, Jiangsu Kaiji Bio-technology Co. Ltd.); 0.25 % Trypsin-EDTA (1×) (Item No. 2120734, Gibco); Penicillin-Streptomycin Mix (Item No. 2257222, Gibco); Fetal Bovine Serum (Item No. 2068801CP, Gibco); Mouse Interleukin 6 (IL-6) Enzyme Immunoassay Kit (Item No. CSB-E04741m, Wuhan Huaqi Biotechnology Co., Ltd.); Mouse Interleukin 6 (IL-6) Enzyme Immunoassay Kit (Item No. E04741m, Wuhan Huaqi Biotechnology Co., Ltd.). Ltd.); Mouse Tumor Necrosis Factor α (TNF-α) Enzyme Immunoassay Kit (Item No. CSB-E04639m, Wuhan Huamei Biological Engineering Co.)

 

1.4 Instruments   

Electronic Analytical Balance (Model: ML204T, Mettler Toledo Guangzhou); Benchtop High-Speed Refrigerated Centrifuge (Model: CT15RE, Hitachi Ltd., Japan); Carbon Dioxide Incubator ［Model: PHCbi MCO-170AICDL-PC, Puhooshi Healthcare Medical Instruments (Shanghai) Co.    (Model: HR1500-IIB2, Qingdao Haier Special Electric Appliances Co., Ltd.); Inverted Biological Microscope (Model: CKX41, OLYMPUS); Ultra-low Temperature Refrigerator (Model: DW-86L728J, Qingdao Haier Special Electric Appliances Co., Ltd.); Ultra-pure Water Apparatus (Model: MERCKMILLIP ORE IQ7000, Nanning City, Guangxi Province, China, Bo-mei Bio-tech Co. Constant temperature water bath (Model: HH-S4, Changzhou Jintan Liangyou Instrument Co., Ltd.); Tissue grinder (Model: 2 ml, Chengdu Shuniu Science and Technology Co., Ltd.); Localized photodynamic therapy instrument (Model: CG-2, Guangzhou Xiangguang Institute of Chinese Medicine Cancer Prevention and Treatment).

 

2 Experimental Methods

2.1 Establishment of animal models   

The experiment was divided into 6 groups: normal group, model group, laser irradiation group, C-PC group, C-PC+laser irradiation group and CY group. There were 10 animals in the normal group and 6 animals in each of the other groups. Except for the normal group, the rest of the groups were modeled according to the method of literature [11]. SW1990 cells were diluted to 5×107/ml with phosphate buffered saline solution (PBS), and then inoculated into the axilla of the right forelimb of nude mice, with 0.2 ml of each nude mouse, to make a human pancreatic adenocarcinoma transplantation model in nude mice, and the whole operation was completed within 30 min. The whole operation was completed within 30 min. The nude mice were kept in a sterile environment, and the model was incubated with a digital vernier caliper every other day starting from day 1.

The longitudinal (a) and transverse (b) diameters of the tumor were measured once, and the modeling was successful when the diameter of the tumor was measured to be about 0.5 cm long.

 

2.2 Therapeutic interventions   

Normal group: no laser irradiation, no medication, and equal volume of drinking water; Model group: no laser irradiation, no medication, and equal volume of drinking water; Laser irradiation group: localized photodynamic therapy device was used to irradiate the tumor area, with the light source 30 cm away from the tumor area of the rats, and the diameter of the light source was 0.5 mm.

2 cm, 15 min each time for 2 weeks; C-PC group: give

500 mg/(kg-d) alginate solution was given by gavage, the volume was 20 ml/kg, and the drug was given continuously for 2 weeks; C-PC+laser irradiation group: 500 mg/(kg-d) alginate solution was given by gavage for 2 h, and then the tumor area was irradiated by local photodynamic therapy instrument, and the irradiated area was irradiated for 15 min each time, and the drug was given for 2 weeks; CY group: 40 mg/(kg-d) cyclophosphamide was given by intraperitoneal injection, and the drug was given by intraperitoneal injection once every other day, and the drug was given continuously for 2 weeks. In the CY group, 40 mg/(kg-d) of cyclophosphamide was given by intraperitoneal injection, and the drug was given by intraperitoneal injection once every other day for 2 consecutive weeks.

 

2.3 Measurement of indicators   

Tumor length (a) and transverse diameter (b) were measured with digital vernier calipers every other day starting from day 1, and mice were weighed at the same time. Mice were weighed 24 hours after the last dose, and the eyes were bled and killed by immersion in 75% sterile alcohol for 5 min. Mice were dissected under aseptic conditions, and the spleens were removed to separate the intradermal from the intact tumors, and the above tissues were peeled off to remove the peripheral membranes, weighed accurately, and the splenic index, tumor volume, and tumor inhibition rate were calculated. The formulas were as follows: Splenic index=spleen mass/body mass (spleen mass in mg, body mass in g); tumor volume=1/2×a×b2 (a is the longitudinal diameter, b is the transverse diameter); tumor inhibition rate (%)=(average tumor weight of the model group - average tumor weight of the experimental group)/average tumor weight of the model group×100%.

 

2.4 Measurement of cytokines

2.4.1 Determination of TNF-α levels in mouse serum   

Whole blood specimens were placed in a refrigerator at 4 ℃ overnight, and then centrifuged at 1,000 g in a 4 ℃ low-temperature centrifuge.

After 15 min, the supernatant was taken and stored at -20 ℃ to avoid repeated freezing and thawing. The serum specimen should be centrifuged again and the supernatant should be taken to determine the TNF-α content according to the instruction of mouse TNF-α enzyme immunoassay kit.

 

2.4.2 Determination of TNF-α levels in mouse spleen cell cultures   

The connective tissue or fat on the spleen tissue was washed away with PBS, 5 ml of L15 complete culture medium was added to a 60 mm2 sterile petri dish, and the spleen was grinded with the flat end of a sterile syringe in a gentle circular motion for 5 times, then filtered through a 200-mesh sieve into a centrifuge tube, centrifuged at 300 g for 10 min, and then washed three times with L15 complete culture medium, and the concentration of the cell suspension was 5×106/ml. The cell suspension was washed three times with L15 complete culture solution, and the concentration of cell suspension was 5×106/ml. The cell supernatant was collected, centrifuged at 1,000 g for 10 min to remove particles and polymers, and the supernatant was extracted and tested for TNF-α activity according to the instruction manual of mouse TNF-α enzyme immunoassay kit.

 

2.4.3 Determination of IL-6 level in mouse serum   

Whole blood specimens were placed in a refrigerator at 4 ℃ overnight and then centrifuged at 1,000 g in a 4 ℃ centrifuge.

After 15 min, the supernatant was extracted and the serum IL-6 level was determined according to the instructions of the mouse IL-6 enzyme immunoassay kit.

 

2.5 Statistical methods   

The experimental data were organized using Excel worksheets, and the data were statistically analyzed using IBM SPSS Statistics 21 software. Measurements were expressed as mean ± standard deviation (x ± s), and comparisons between groups were made using one-way analysis of variance (ANOVA), with P<0.05 being statistically significant.

 

3 Results

3.1 General Observations   

Mice in the normal group had a good mental state and smooth skin without roughness and wrinkles; mice in the model group became tumors, had reduced activities, were depressed and lethargic, lost weight, and had rough and wrinkled skin surfaces; mice in the CY group were affected by the chemotherapeutic drug cyclophosphamide, were lethargic, had little activity, and most of them curled up with their backs in the shape of a bow when they were not active; mice in the C-PC group, the laser irradiation group and the C-PC+ laser irradiation group were more active in terms of mental state and activities. The C-PC, laser irradiation and C-PC+laser irradiation groups were more active. The tumor formation rate of the animals was 100%, and there was no death of mice during the whole animal feeding experiment.

 

3.2 Comparison of tumor weight, tumor volume and tumor inhibition rate of mice in each group is shown in Table 1 Fig. 1.

The results showed that compared with the model group, the tumor weight of the C-PC group was significantly reduced (P<0.05); the tumor weight and tumor volume of the CY group and the C-PC+laser irradiation group were significantly reduced (P<0.05 or P<0.01); and the laser irradiation group did not have any significant effect on the tumor weight and tumor volume. The tumor suppression rates of the laser irradiation group, C-PC group, and C-PC+laser irradiation group were (39.42±8.47)%, (42.85±12.15)%, and (74.80±11.85)%, respectively, and the tumor suppression rate of the C-PC+laser irradiation group was comparable to that of the CY group (78.58±8.87%) (P>0.05).

 

3.3 Comparison of splenic weight and splenic index of mice in each group is shown in Table 2.

Compared with the normal group, the spleen weight and spleen index of the model group were significantly reduced (P<0.05); compared with the model group, the laser irradiation group, the C-PC group and the C-PC+laser irradiation group significantly increased the spleen weight and spleen index (P<0.05 or P<0.01), while the spleen weight and spleen index of the CY group mice were significantly decreased (P<0.05). The results showed that laser irradiation or phycocyanin treatment alone had immune-enhancing effects, and the combination of the two had more significant effects.

 

3.4 Comparison of TNF-α and IL-6 levels of mice in each group is shown in Table 3.

The laser irradiation group promoted IL-6 levels (P<0.05), but had no significant effect on TNF-α levels (P>0.05).

 

4 Discussion

Pancreatic adenocarcinoma (PDA) is a highly malignant tumor of the digestive system, the incidence of which is increasing year by year, and the rate of early diagnosis is not high, and it is one of the cancers with a poor surgical prognosis [12-13]. Under normal conditions, the body's immune system is in a state of mutual balance, but when a tumor develops, the body's immune function decreases and the balance is disrupted. Therefore, the occurrence and development of tumors are closely related to the immune system [14]. It has been proved that photodynamic therapy can induce specific and nonspecific immune responses, thus improving the effect of photodynamic-mediated antitumor [15], and the combined application of photodynamic therapy and antitumor immunotherapy has significant synergistic antitumor effects [16-18].

 

In this experiment, nude mice were used as experimental subjects, and human pancreatic cancer SW1990 cells were subcutaneously inoculated in the axilla of the right forelimb of nude mice. Since the tumor cells were heterologously inoculated in the subcutaneous area of animals, the tumor latent growth cycle was longer, and it generally took about 7-14 d to reach the required tumor volume in the experimental study, but the tumorigenicity rate was better, and it was as high as 100%. The results of in vivo experiments showed that laser irradiation, phycocyanin treatment and phycocyanin combined with laser irradiation could reduce the tumor weight and volume, and the tumor inhibition rates were (39.42±8.47)%, (42.85±12.15)% and (74.80±11.85)%, respectively, and that of phycocyanin combined with laser irradiation was similar to that of CY (78.58±8.87)%. The tumor inhibition rate of phycocyanin combined with laser irradiation was similar to that of the CY group (78.58±8.87)%, indicating that the combination of phycocyanin and photodynamic therapy has a good effect on inhibiting tumor growth. The tumor inhibition rate of the laser irradiation group reached 30%, which was also considered to have a certain tumor inhibition effect [19].

 

The results of in vivo experiments showed that the spleen weight and spleen index of immune organs in the model group were significantly reduced. After laser irradiation and phycocyanin treatment, the spleen weight and index of mice increased significantly, indicating that laser irradiation and phycocyanin treatment can promote the growth and development of the mouse spleen, and the increase in spleen weight and index of mice irradiated with phycocyanin combined with laser irradiation was more significant, and the mechanism of this may be to enhance the immune activity of mice by inducing a proliferative response of the activated cells of the immune organs [20].

 

Tumor necrosis factor (TNF) is the strongest anti-tumor cytokine discovered so far [21], with anti-tumor, anti-virus and immunomodulatory biological activities [22]. It has been shown that TNF-α can directly induce apoptosis of tumor cells in vivo, and can exert antitumor and immunomodulatory effects through multiple pathways [23]. Interleukin-6 (IL-6) is a lymphokine produced by activated T cells, which enables B cell precursors to become antibody-producing cells, and is directly related to inflammatory diseases and the degree of infection.

 

Early studies have shown that IL-6 can play an anti-tumor role by inducing immune responses to directly or indirectly inhibit the growth of tumor cells [24]. The results of this experiment showed that the expression of TNF-α and IL-6 was higher than that of the model group after treatment with phycocyanin and laser irradiation, and the combined effect of the two was more significant. With the prolongation of disease and deterioration of malignant tumor patients, the function of immune system often decreases, and the decrease of immune system function is more obvious with the increase of the number of radiotherapy and chemotherapy. Phycocyanin and laser irradiation can significantly increase the levels of TNF-α and IL-6 in mice with cancer, and regulate the immunity effect of the body, which may be one of the anti-tumor mechanisms.

 

In conclusion, alginate can effectively inhibit the growth of tumor tissues in vivo, and the effect of photodynamic therapy mediated by alginate is even more significant in the treatment of SW1990 tumors in mice, which can increase the spleen index of immune organs and promote the expression of TNF-α and IL-6 in mice with cancer. This suggests that algin combined with photodynamic therapy may regulate the immune function of the body to inhibit tumor growth.

 

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