Effect of New Water-Soluble Dendritic Phthalocyanines on Human Colorectal and Liver Cancer Cell Lines

Human hepatocellular carcinoma (HepG2) cells and colorectal adenocarcinoma (DLD-1) cells were treated with the synthesized water soluble phthalocyanine derivatives to understand the effect of the compounds both on colorectal and liver cancer cells. The compounds inhibited cell proliferation and displayed cytotoxic effect on these cancer cell lines however; the effect of the compounds on healthy control fibroblast cell line was comparatively lower. The compounds can be employed for cancer treatment as anticancer agents.


Introduction
Colorectal cancer is one of the most common causes of deaths in the world.The most frequent metastastatic place of the colorectal cancer is the liver.Major cause of the deaths in patients with colorectal cancer is the liver metastasis and almost half of colorectal cancer patients are diagnosed with the liver metastases eventually.Treatment of colorectal cancer liver metastasis includes a combination of surgery, chemotheraphy, and radiotheraphy.
5-fluorouracil, leucovorin, kapesitabin, irinotecan, oxaliplatin, and their combinations are used for treatment of colorectal cancer liver metastasis.These chemotherapeutics inhibit DNA synthesis in cancer cells and prevent DNA replication and transcription, causing cell death.However, their low selectivity causes some limitations and side effects.To compensate selectivity and limitation problems, monoclonal antibodies (cetuximab, bevasizumab, panitumomab) are used in combination with these drugs in colorectal cancer liver metastasis treatment.Therefore, researchers and global pharmaceutical companies focus on the development of efficient colorectal and liver cancer drugs [1,2].
On the other hand, it is important to synthesis water soluble phthalocyanine compounds for biomedical applications [25,26].In the literature, different water soluble dendritic phthalocyanine compounds have been synthesized and their photophysical [27,28,29], spectroscopic [30,31], electron transfer processes [32,33], electrical and CO2 sensing properties [34] have been investigated.Therefore, synthesis of new water soluble dendritic phthalocyanine compounds and investigation of their anticancer activities could be worthwhile.
In this study, water soluble dendritic metal-free 3a and zinc phthalocyanine 3b compounds were synthesized and characterized.The potential of anticancer activity of 3a and 3b was evaluated in human colorectal and liver cancer cell lines.Furthermore, the cytotoxicity of compounds was determined in healthy fibroblast cell line.Experimental data indicated that these compounds displayed minimal activity against healthy fibroblast cells but effectively toxicated the cancer cell lines.The study also aimed to offer new potential therapeutic agents for the treatment of human colorectal cancer liver metastasis.

Compounds 1a and 1b
The solution of triethyl methanetricarboxylate sodium salt (NaC(CO2C2H5)3) was prepared with reaction of metallic Na (12.30 mg, 0.54 mmol) and triethyl methanetricarboxylate (0.23 mL, 1.07 mmol) in dry ethanol (6 mL) at room temperature.Tetrakis [ (2-trimethylaminoethylsulfanyl) phthalocyanine] tetraiodide (200.0mg,0.13mmol) or tetrakis[(2trimethylaminoethylsulfanyl) phthalocyaninatozinc (II)]tetraiodide (203.0 mg, 0.13 mmol) was added to the prepared solution of NaC(CO2C2H5)3.The mixture was refluxed for 3h.After cooled down to room temperature, 2.0 mL of 40% NaOH solution was added to reaction medium, stirred for 10 min, 2.0 mL H2O was added to it and continued to reflux for 4h.The reaction mixture was concentrated by evaporation of ethanol and the dark green aqueous solution was extracted with ether.The product was precipitated by acidify of the aqueous phase with diluted hydrochloric acid.The green solid was filtered off, washed with H2O (3x 20 mL) and acetone (2x 3 mL) and was dried in vacuum.The green solids 1a and 1b were soluble in DMSO and DMF.

Compounds 2a and 2b
Mixtures of 1a (100.0 mg, 0.06 mmol) and H2NC(CH2OH)3 (103.0 mg, 0.84 mmol) in DMSO (3 mL) were heated at the presence of K2CO3 (332.0 mg, 2.4 mmol) at 180 o C for 24h.After cooled down to room temperature, the product was precipitated in ether, filtered off and dried.The crude residue was washed with H2O (3x 5 mL), ethanol (2x 3 mL) and acetone (3x 3 mL), respectively.2b was synthesized using a similar procedure to 2a.The dark green solids 2a and 2b were soluble in DMSO and DMF.

Compounds 3a and 3b
Phthalocyanine 2a (100.0 mg, 0.039 mmol) or 2b (100.0 mg, 0.038 mmol) were added to NaOH solution (30%, 5 mL).This mixture was refluxed until a clear solution was formed.At the end of the reaction, the dark green reaction solution was precipitated in methanol (100 mL) and filtered, respectively.The resulting solid was dissolved in H2O and the solution was again precipitated with methanol until neutral.The product washed with methanol, acetone and dried in vacuum etuv at 80 o C.
The green solids 3a and 3b were soluble in H2O and DMSO at room temperature.

Cytotoxicity and anticancer activity tests
Cytotoxicity and anticancer activity assays were performed using colorimetric XTT cell proliferation test [37,38].The cells was cultivated in a flat 96-well culture plate (10x10 4 cells/well was seeded in the 100 µL culture medium).Each plate included a blank containing complete medium without cells.Compounds 3a and 3b were diluted to obtain five different concentrations ranging from 10 -4 to 10 -8 M. The plates were incubated in a humidified CO2 incubator at 37°C throughout 24 h and 48 h.At the end of these incubation periods, 3a and 3b compounds were removed and the wells were washed with steryl PBS three times.50 μl of the XTT solution was added to each well and the plate was incubated in an incubator for four hours.Wells were gently shaked to evenly distribute the dye and absorbance of the samples against a background control as a blank with ELISA reader at a wavelength of 450 nm was measured.

Statistical analysis
Differences in the mean values of the measured activities were statistically evaluated using the SPSS 17.0 program.(Univariate Variance Analyses and Pearson Correlation).Probability values of p < 0.05 were considered to be significant.
In the 1 H-NMR spectra of water soluble dendritic phthalocyanine compounds 3a and 3b were not observed to -OH proton peak, this result shows that all of the -OH groups in the compounds 2a and 2b have been hydrolyzed.
The UV-vis spectra of metal-free dendritic phthalocyanine compounds 1a, 2a and 3a in DMSO showed two intense absorptions between 710 nm and 682 nm.The UV-vis spectra of dendritic zinc phthalocyanine compounds 1b, 2b and 3b in DMSO give a peak at around 688 nm, 690 nm and 692 nm, respectively [48].In addition, B-band of componds 1-3 appeared at between 342-365 nm (Figure 2).

Cytotoxicity and anticancer activity of 3a and 3b
Cytotoxic effects of compounds were analysed in L929 fibroblasts by XTT assay performed in cultures exposed for 24h and 48h.Fibroblast cells were selected because they are widely distributed in many types of tissues.Especially, L929 cell line is recommended in many biological experiments such as material biocompatibility testing, drug cytotoxicity testing and cell biology studies.Moreover, the cells can be cultured easily and have a proper doubling time of 24 h.According to the results, water soluble dendritic phthalocyanines 3a and 3b showed low cytotoxicity at the three extreme concentrations (10 - 4 , 10 -5 and 10 -6 M).Furthermore, increasing incubation time did not induce further cytotoxic effects on L929 cell line dramatically.
Antitumor properties of 3a and 3b were tested in vitro against two human cancer cell lines (DLD-1 and HepG2) derived from colorectal and liver.According to the results, 3a and 3b showed great anticancer potency and were able to kill both DLD-1 and HepG2 cells at 10 -4 M and 10 -5 M. Furthermore, cell viability was found to decrease with incubation time.Especially, the strongest anticancer activity was found for 3b, and also addition of 10 -4 M 3b led to 19,59% and 9,95% higher toxicity than 10 -4 M 3a for colorectal and liver cancer cells respectively after 24 h, and these ratios were determined as 17,34% and 10% after 48 h.Furthermore, 10 -5 M 3b caused 7,45% and 4,13%, 13% and 11,81% higher toxicity than 10 -5 M 3a for colorectal and liver cancer cells after 24 h and 48 h, respectively (Table 1) (Figure 3).In current studies, anticancer activities of anionic metal-free 3a and Zn(II) 3b phthalocyanine dendrimers were evaluated.It has been observed that these compounds have potential for development as cancer drugs.
In our study, we observed antitumor activity of anionic phthalocyanine-based dendrimers in colorectal and liver cancer cells.The presence of phthalocyanine increased antitumor activity as well as participation of Zn in phthalocyanine core triggers this activity.Especially zinc phthalocyanine compounds are observed to be more effective in biomedical applications in the literature [49,50].It is known that, dendrimers provide better opportunity for drug delivery due to their high functionality, low polydispersity, and three-dimensional architecture.Apart from these, dendrimers protect drugs from biological degradation and increase their stability [51,52].Our novel metal-free 3a and zinc(II) 3b phthalocyanine dendrimers may offer new compounds for using site specific drug delivery.

Discussion and Conclusion
The water soluble dendritic metal-free and zinc(II) phthalocyanines were designed for highly soluble and stable agents for colorectal liver metastases treatment.We studied anticancer activity of water soluble dendritic phthalocyanines 3a and 3b in colorectal and liver cancer cell lines and also the cytotoxicity of the compounds in healthy fibroblast cell line.The designed compounds may use as anticancer agent to destroy cancer cells.
The presented results confirm that new water soluble dendritic metal-free 3a and zinc phthalocyanines 3b may be utilized for colorectal and liver metastases treatment.Moreover, these new compounds have great potential for further investigations and applications.

Figure 3 .
% Toxicity observed in different cell lines of compounds 3a and 3b (a) after 24 hours of incubation (b) after 48 hours of incubation.

Table 1 .
Anticancer activity of 3a and 3b as % cell viability after 24h and 48h.