B16 Melanoma Cancer Research Articles

Sodium copper chlorophyllin-loaded chitosan nanoparticle-based photodynamic therapy for B16 melanoma cancer cells.

Melanoma is one of the most aggressive and fatal skin cancers owing to its ability to metastasize and develop resistance to chemotherapy. Photodynamic therapy (PDT) is a minimally noninvasive treatment modality comprising photosensitizers (PSs), light sources, and endogenous molecular oxygen that exert a localized cytotoxic effect on cancer cells. The current study explores the therapeutic potential of sodium copper chlorophyllin-loaded chitosan nanoparticles (CH-SCC NPs) along with handheld laser-based PDT on B16 cancer cells. A modified chlorophyll derivative identified as sodium copper chlorophyllin (SCC) is a dietary supplement that has anticancer properties. Herein, we have synthesized CH-SCC NPs using the ionic gelation method to enhance the PS's bioavailability and efficiency. Chitosan nanoparticles exhibited high biocompatibility in a normal cell line L929, zebrafish, and chick embryos, and were successfully employed to deliver the SCC to cancer cells. CH-SCC NPs showed an enhanced PDT effect that killed approximately 80%-85% of B16 cells. CH-SCC NPs in combination with a handheld portable laser source showed significant therapeutic potential against the B16 skin cancer cell line. The experimental findings further strengthen our device-repurposing strategy, which suggests that SCC nanoformulations along with handheld laser can be a suitable treatment for skin cancer even in remote areas where power source and treatment cost can be a limitation.

Melanin-Ce6-loaded polydopamine nanoparticles-based enhanced phototherapy for B16 melanoma cancer cells

Melanoma is one of the most aggressive and lethal types of cancer owing to its metastatic propensity and chemoresistance property. An alternative therapeutic option is photodynamic and photothermal therapies (PDT/PTT), which employ near-infrared (NIR) light to generate heat and reactive oxygen species (ROS). As per previous reports, Melanin (Mel), and its synthetic analogs (i.e. polydopamine nanoparticles) can induce NIR light-mediated heat energy, thereby selectively targeting and ameliorating cancer cells. Similarly, chlorin e6 (Ce6) also has high ROS generation ability and antitumor activity against various types of cancer. Based on this tenet, In the current study, we have encapsulated Mel-Ce6 in a polydopamine (PDA) nanocarrier (MCP NPs) synthesized by the oxidation polymerization method. The hydrodynamic diameter of the synthesized spherical MCP NPs was 139 ± 10 nm. The MCP NPs, upon irradiation with NIR 690 nm laser for 6 min, showed photothermal efficacy of more than 50 °C. Moreover, the red fluorescence in the MCP NPs due to Ce6 can be leveraged for diagnostic purposes. Further, the MCP NPs exhibited considerable biocompatibility with the L929 cell line and exerted nearly 70% ROS-mediated cytotoxicity on the B16 melanoma cell line after the laser irradiation. Thus, the prepared MCP NPs could be a promising theranostic agent for treating the B16 melanoma cancer.

PD-1 Blockade on Tumor Microenvironment-Resident ILC2s Promotes TNF-α Production and Restricts Progression of Metastatic Melanoma.

While pulmonary ILC2s represent one of the major tissue-resident innate lymphoid cell populations at steady state and are key drivers of cytokine secretion in their occupational niche, their role in pulmonary cancer progression remains unclear. As the programmed cell death protein-1 (PD-1) plays a major role in cancer immunotherapy and immunoregulatory properties, here we investigate the specific effect of PD-1 inhibition on ILC2s during pulmonary B16 melanoma cancer metastasis. We demonstrate that PD-1 inhibition on ILC2s suppresses B16 tumor growth. Further, PD-1 inhibition upregulates pulmonary ILC2-derived TNF-α production, a cytotoxic cytokine that directly induces cell death in B16 cells, independent of adaptive immunity. Together, these results highlight the importance of ILC2s and their anti-tumor role in pulmonary B16 cancer progression during PD-1 inhibitory immunotherapy.

B16 Melanoma Cancer Cells with Higher Metastatic Potential are More Deformable at a Whole-Cell Level.

Metastasis is a process in which cancer cells spread from the primary focus site to various other organ sites. Many studies have suggested that reduced stiffness would facilitate passing through extracellular matrix when cancer cells instigate a metastatic process. Here we investigated the compressive properties of melanoma cancer cells with different metastatic potentials at the whole-cell level. Differences in their compressive properties were analyzed by examining actin filament structure and actin-related gene expression. Compressive tests were carried out for two metastatic B16 melanoma variants (B16-F1 and B16-F10) to characterize global compressive properties of cancer cells. RNA-seq analysis and fluorescence microscopic imaging were performed to clarify contribution of actin filaments to the global compressive properties. RNA-seq analysis and fluorescence microscopic imaging revealed the undeveloped structure of actin filaments in B16-F10 cells. The Young's modulus of B16-F10 cells was significantly lower than that of B16-F1 cells. Disruption of the actin filaments in B16-F1 cells reduced the Young's modulus to the same level as that of B16-F10 cells, while the Young's modulus in B16-F10 cells remained the same regardless of the disruption. In B16 melanoma cancer cell lines, cells with higher metastatic potential were more deformable at the whole-cell level with undeveloped actin filament structure, even when highly deformed. These results imply that invasive cancer cells may gain the ability to inhibit actin filament development. The online version contains supplementary material available at (10.1007/s12195-021-00677-w).

Cytotoxic Activity of Ethyl-para-methoxycinnamate from Kaempferia galanga L. on A549 Lung Cancer and B16 Melanoma Cancer Cells

Kaempferia galanga L. belongs to the family of Zingiberaceae, an endangered medicinal plant with pharmacology activities. Ethyl-p-methoxycinnamate (EPMC) is an essential phytoconstituent of K. galanga rhizomes. Several studies have reported that EPMC has anticancer activities in several cancer cells, including CL-6 gallbladder cancer cells, HepG2liver cancer cells, MCF-7 breast cancer cells, and Raji lymphoma cancer cells. However, studies on A549 lung cancer and B16 melanoma cancer cells have not been reported. This study aimed to determine the anticancer activity of EPMC against A549 lung cancer and B16 melanoma cancer cells. EPMC was obtained by extraction using n-hexane, then recrystallized with chloroform. The isolate was then analyzed by thin-layer chromatography (TLC), and the structure was characterized by Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopy. Cytotoxic activity was determined under Presto Blue assay. Based on the result, EPMC from K. galanga showed the cytotoxic effect on B16 cells with an IC50 value of 97.09 μg/mL, whereas EPMC showed no significant cytotoxic effect on A549 with an IC50 value of 1407.75 μg/mL. It was concluded that EPMC has potential cytotoxic on B16 melanoma cancer cells, but it showed inactive activity against A549 lung cancer cells. Further molecular mechanism underlying EPMC cytotoxic activity needs to be conducted.

Anti-proliferative Activity of Some Oleanolic Acid Derivatives with Potent Topoisomerase Inhibitory Activity on B16 Melanoma Cells

Our previous study included the semisynthetic reactions on oleanolic acid, a common wood-derived oleanane-type triterpene. Ten rationally designed derivatives of oleanolic acid were synthesized based on docking studies and tested for their topoisomerase I and IIα inhibitory activity. Semisynthetic reactions targeted C-3, C-12, C-13 and C-17. Some of these compounds act as dual inhibitors for both topoisomerase I and IIα giving new anticancer agents. The cytotoxic activity of these compounds on B16 melanoma cancer cells was evaluated. Results showed that most of these compounds have a higher cytotoxic activity on B16 melanoma cells.

Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments.

In the context of research for new cytotoxic compounds, obtaining bioactive molecules from renewable sources remain a big challenge. Microorganisms and more specifically Actinobacteria from original sources are well known for their biotechnological potential and are hotspots for the discovery of new bioactive compounds. The strain DP94 studied here had shown an interesting cytotoxic activity of its culture broth (HaCaT: IC50 = 8.0 ± 1.5 μg/mL; B16: IC50 = 4.6 ± 1.8 μg/mL), which could not been explained by the compounds isolated in a previous work. The increase of the cytotoxic activity of extracts was investigated, based on a Taguchi L9 orthogonal array design, after DP94 culture in TY medium using two different vessels (bioreactor or Erlenmeyer flasks). Various culture parameters such as temperature, pH and inoculum ratio (%) were studied. For experiments conducted in a bioreactor, stirring speed was included as an additional parameter. Significant differences in the cytotoxic activities of different extracts on B16 melanoma cancer cell lines, highlighted the influence of culture temperature on the production of cytotoxic compound(s) using a bioreactor. A culture in Erlenmeyer flasks was also performed and afforded an increase of the production of the active compounds. The best conditions for the highest cytotoxicity (IC50 on B16: 6 ± 0.5 μg/mL) and the highest yield (202.0 mg/L) were identified as: pH 6, temperature 37°C and 5% inoculum.

Ratiometric and light-up near-infrared fluorescent DCM-based probe for real-time monitoring endogenous tyrosinase activity

Tyrosinase (TYR), which is regarded as a significant biomarker of melanoma cancer, plays a crucial but incompletely understood role in cellular biochemistry and etiology. In this work, we report a TYR activatable NIR fluorescent probe DCM-1, which is composed of dicyanomethylene-4H-pyran (DCM) unit as the NIR fluorescence reporter, amide bond specific for TYR cleavage, and phenol group as the triggered moiety. The activation of DCM-1 by endogenous TYR can induce a remarkable fluorescence red shift from 548 to 660 nm, allowing for ratiometric and light-up NIR response to real-time track TYR activity. Besides, DCM-1 exhibits extraordinary features, such as excellent sensitivity, high selectivity, and high photostability. In vitro experiments by confocal laser scanning microscopy reveals that the spatial-temporal information of TYR in B16 melanoma cancer cells can be achieved by the probe DCM-1. Thus, this enzyme-activatable NIR probe can be a powerful tool for elucidating the roles of TYR in biological systems.

PO-364 Effect of exercise and immunotherapy on tumour immunogenicity and growth

IntroductionSeveral preclinical studies have shown that exercise can reduce the tumour growth across a range of tumour models. We recently demonstrated that this exercise-dependent suppression of tumour growth was due to an epinephrine-dependent mobilisation and IL-6-driven activation of cytotoxic cells. In line with this, exercise markedly increased intratumoral immune cell infiltration, thus promoting an immune-stimulatory intratumoral environment. Based on these original findings, we hypothesised that exercise may enhance the effect of immune checkpoint blockade treatment given the exercise-dependent increased intratumoral immune cell infiltration.Material and methodsWe used C57Black/C mice randomised to cages with or without running wheels (EX). After 4 weeks of running (with a running distance ranging from 1.8 km to 5.4 km per mice per day), the mice were inoculated with B16 melanoma cancer cells. In addition, a subset of mice was treated with αPD-L1 antibodies (100 µg per mouse three times per week starting on day 4 after tumour cell injection).Results and discussionsFirst, we observed that voluntary wheel running increased intratumoral expression of the immune checkpoint molecules, PD-L1, B7.1 and B7.2. In parallel, voluntary wheel running increased intratumoral infiltration of cytotoxic NK and T cells, and thus intratumoral expression of the receptors, PD-1 and CD28. Next, we tested the combination therapy of exercise and αPD-L1 antibody treatment. In this setup, wheel running had an overall suppressive effect (p=0.01, 2-way ANOVA). Post hoc analysis showed that αPD-L1 +EX reduced tumour growth by 82.6%, while EX alone reduced tumour growth by 72% with no significant difference between the two interventions. qPCR analysis of these tumours revealed that combination therapy further increased the expression of immune cell markers and immune check point molecules.ConclusionIn conclusion, our results show that exercise increases the expression of immune checkpoint markers and therefore, could be a good combination partner for immune checkpoint blockade treatment. However, more studies are needed to show an effect of this combination therapy on tumour growth.

Glycolipid stimulation of invariant NKT cells mobilizes precursors of mature NK cells and potentiates their participation in immune surveillance against metastatic cancer.

Abstract Precursors of mature natural killer (pre-mNK) cells, initially called IFN-producing killer dendritic cells, have been recently characterized as a novel intermediate in NK cell differentiation. Typified by a unique B220+NK1.1+CD11c+MHCII+ phenotype, pre-mNK cells exhibit prolific anti-tumor cytotoxicity while retaining the ability to present antigens thereby activating neighbouring T cells. Invariant NKT (iNKT) cells are another population highlighted for their potential in anti-cancer immunity. They are activated very early and play an important role in transactivating downstream effectors, such as NK cells. The extent to which iNKT cells can activate pre-mNK cells is unknown. We hypothesized that iNKT cells are able to activate pre-mNK cells and potentiate their anti-tumor properties. Wildtype C57BL/6 mice were injected i.p. with the iNKT cell superagonist, α-galactosylceramide (αGC), and various lymphoid tissues were harvested after 5 days for phenotyping or cytotoxicity assays. We have shown, for the first time that iNKT cell activation via αGC results in robust expansion of pre-mNK cells in the spleen, lung and most notably, in the liver. These cells exhibited cytotoxic activity against YAC-1 thymoma and B16 melanoma cancer cells via the granule exocytosis pathway and significantly contributed to overall NK cytotoxic activity in vivo. The anti-cancer responses due to αGC activation have been well documented; however, the contribution of pre-mNK cells in these responses have never been shown. Our findings demonstrate that pre-mNK cells rapidly expand due to iNKT activation while retaining their capacity to kill tumor targets. This may suggest a novel mechanism of targeting pre-mNK cells in anti-cancer therapies.

Separate roles of IL-6 and oncostatin M in mouse macrophage polarization in vitro and in vivo.

Arginase-1 (Arg-1)-expressing M2-like macrophages are associated with Th2-skewed immune responses, allergic airway pathology, ectopic B16 melanoma cancer growth in murine models, and can be induced by Oncostatin M (OSM) transient overexpression in vivo. Here, we compare OSM to the gp130-cytokine IL-6 in mediating macrophage polarization, and find that IL-6 overexpression alone (Ad vector, AdIL-6) did not induce Arg-1 protein in mouse lungs at day 7, nor ectopic melanoma tumor growth at day 14, in contrast to overexpression of OSM (AdOSM). AdOSM elevated levels of IL-4, IL-5 and IL-13 in bronchoalveolar lavage fluid, whereas AdIL-6 did not. Bone marrow-derived macrophages respond with Arg-1 enzymatic activity to M2 stimuli (IL-4/IL-13), which was further elevated in combination with IL-6 stimulation; however, OSM or LIF had no detectable activity in vitro. Arg-1 mRNA expression induced by AdOSM was attenuated in IL-6-/- and STAT6-/- mice, suggesting requirements for both IL-6 and IL-4/IL-13 signaling in vivo. Ectopic B16 tumor burden was also reduced in IL-6-/- mice. Thus, OSM induces Arg-1+ macrophage accumulation indirectly through elevation of Th2 cytokines and IL-6 in vivo, whereas IL-6 acts directly on macrophages but requires a Th2 microenvironment, demonstrating distinct roles for OSM and IL-6 in M2 macrophage polarization.

Novel peptide MT23 for potent penetrating and selective targeting in mouse melanoma cancer cells

Cell-penetrating peptides (CPPs) have a great potential for intracellular delivery of cell-impermeable biological macromolecules in clinical therapy. However, their lack of cell and tissue specificity remains the primary limitation for their clinical development as drug delivery vehicles. In this study, based on phage display and an in silico approach, we found a novel CPP-MT23 with mouse melanoma cell specificity, it can only enter B16 melanoma cancer cells and without any cytotoxicity, Moreover, MT23 showed higher penetration efficiency based on fluorescence microcopy and quantitative assay, and it has capability for mediating functional Apoptin into cells in vitro or in vivo. Moreover, MT23-Apoptin can significantly inhibit tumor growth and induce the cell apoptosis in B16 tumor bearing mice. To sum up, all the results implicated that MT23 has the potential to deliver exogenous therapeutic proteins for further use and it also expected to lay the foundation for developing human melanoma cancer cell specific CPP.

Abstract 4610: Towards understanding the cellular uptake patterns of nano-particles among different immune cell lines

Abstract Today with the privilege of being able to synthesize extremely small particles in the nano-range and make use of nanoscience and nanotechnology, we can reach superior properties than the bulk scale. Nanoparticles have their own physicochemical properties that make them promising in the field of cancer immunotherapy in form of drug delivery, diagnostic and theranostic modalities. Manipulation of the immune response by therapeutic intervention is becoming of great interest owing to the significant role of immunity in the general health and disease control. APCs like Dendritic cells and macrophages are important targets for the particulate delivery system due to their ability to trigger cascade of events on both levels cellular and humoral immune response specially DCs. Successful targeting of DCs and macrophages will have a great impact on T-cell activation and priming. The design of successful particulate system that can elicit preferential targeting towards specific immune cell in the tumor microenvironment is becoming crucial. This might represent a novel avenue for enhancing the antitumor immunity. Liposomes are made of phospholipid bilayer with a composite nature. Their properties are highly linked to their physicochemical characteristics such as size, surface charge and composition. These physicochemical properties can trigger certain immune response.The hypothesis here whether passive targeting via particulate system can offer a platform for preferential targeting for critical immune cells residing in spleen and tumor. Moreover, test whether there will be differences in uptake according to certain preference towards surface charge in specific time frame. In the current study, three sets of fluorescently labelled nano-liposomes were engineered as a model for different surface charges, the cationic DOTAP NP, anionic DOPG NP and near neutral DOPC NP with mean diameter of ~ 200 nm. Physical stability of the NPs was evaluated by monitoring the changes in size and zeta potential. B16 melanoma cancer model was induced subcutaneously in C57BL/6 black mice, divided into four groups each of five mice. CD11c Dendritic Cells (DCs), CD11b macrophages, CD90.2 T-cells and CD49b Natural Killer (NK) cells were isolated from the tumors and spleens of each group. The three sets of NPs were tested against the isolated cell lines. The cellular uptake (internalization) was assessed by normalizing the fluorescence of the cells against their protein concentration, then all samples were acquired to flow cytometry, and shifts in fluorescence histograms on horizontal axis were monitored against PE channel on the vertical axis. Results reveal the presence of preferential internalization of specific surface charge over others in some cell lines in different time frames. For the first time differences in the internalization pattern are reported in the same immune cell line isolated from two different contexts tumor and spleen. Note: This abstract was not presented at the meeting. Citation Format: Noha Ismail, Ashish Kulkarni, Siva Kumar, Vineeth krishna, Shiladitya Sengupta. Towards understanding the cellular uptake patterns of nano-particles among different immune cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4610. doi:10.1158/1538-7445.AM2017-4610

Abstract 4274: Anticancer mechanisms of a small amphipathic molecule, LTX-401, against B16 melanoma cancer cells

Abstract In the present study we investigated the mechanism behind the killing of B16F1 murine melanoma cells by the small amphipathic β(2,2)-amino acid derivative, LTX-401 (Mw<500Da). In vitro, LTX-401 induced lytic cell death and the release of Danger-Associated-Molecular-Pattern Molecules(DAMPs) such as ATP and HMGB-1. In addition, the cells released pro-Cathepsin-B and cytochrome-c following LTX-401 treatment. Flow cytometry and confocal microscopy studies revealed reduced signal from the lysomal dye Lysotracker-DND-26, indicating loss of lysomal integrity. Furthermore, flow cytometry analysis showed that the mitochondrial membrane potential was unaffected in the LTX-401 treated cells. Transmission electron microscopy of LTX-401 treated cells showed little or no ultra structural changes in the mitochondria. The in vivo antitumor effect of LTX-401 against intradermally established B16F1 melanoma was assessed in syngeneic mice. Intratumoral administration of the molecule caused rapid tumor necrosis and complete tumor regression in the majority of the animals treated. In conclusion, these findings demonstrate that LTX-401 has the ability to cause immunogenic cell death in cancer cells and induce complete tumor regression in solid tumors and thus might have a potential as an immunotherapeutic agent. Citation Format: Liv-Marie Eike, Brynjar Mauseth, Ketil Camilio, Oystein Rekdal, Baldur Sveinbjornsson. Anticancer mechanisms of a small amphipathic molecule, LTX-401, against B16 melanoma cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4274. doi:10.1158/1538-7445.AM2015-4274

The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit

IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

In vitro and in vivo biological evaluation of new 4,5-disubstituted 1,2,3-triazoles as cis-constrained analogs of combretastatin A4

To find new and better antivascular agents for cancer therapy, a series of combretastatin A4 (CA4) analogs were prepared from 1,3-diaryl-2-nitroprop-1-enes (6–12) obtained in a two-step synthesis from appropriate arylaldehydes and 2-aryl-1-nitroethanes (4 or 5). Treatment of these 1,3-diaryl-2-nitroprop-1-enes 6–12 by sodium azide in DMSO yielded the targeted compounds. The synthesized 1,2,3-triazoles disubstituted in 4- and 5-positions by one benzyl group and one aryl nucleus have also been tested for biological activities involved in antivascular action. It was found that several new compounds exhibited interesting biological activities in the nanomolar or low micromolar range, in terms of rounding up of endothelial cells, inhibition of tubulin polymerization, and cytotoxicity on B16 melanoma cancer cells. In silico docking studies of 11 and 19 within the active site of tubulin were also carried out in order to rationalize the inhibitory properties of these compounds and further understand their inhibition mechanism. In vivo evaluation of compounds 11 and 19 in mice bearing colon 26 carcinoma indicated modest anticancer activity.

IFN-α-Induced Murine B16 Melanoma Cancer Vaccine Cells: Induction and Accumulation of Cell-Associated IL-15

Many components of the class I antigen-processing pathway are thought to be regulated solely by interferon-gamma (IFN-gamma). Herein, we report type I IFN-mediated induction of proteasome activator (PA28) subunits alpha and beta, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and leucine aminopeptidase (LAP). This mechanism was initiated by either synthetic RNA (poly(I-C)) or by hepatitis C virus (HCV) RNA-mediated induction of type I IFN and abrogated by blocking of type I IFN. In serial liver biopsies of chimpanzees with acute HCV infection, increases in PA28 subunit and aminopeptidase mRNA levels correlated with intrahepatic type I IFN responses and preceded intrahepatic IFN-gamma responses by several weeks. Thus, viral RNA-induced type I IFN regulates the antigen-processing machinery early during viral infection and prior to IFN-gamma response. This mechanism may contribute to the high effectiveness of type I IFN-based therapies if administered early during acute HCV infection.

Preparation and in vitro photodynamic activities of novel axially substituted silicon (IV) phthalocyanines and their bovine serum albumin conjugates

Two novel axially substituted phthalocyanines, namely bis(4-(4-acetylpiperazine)phenoxy)phthalocyaninatosilicon (IV) ( 1) and its N-methylated derivative 2, have been synthesized. The dicationic phthalocyanine 2 is non-aggregated in water and exhibits good photophysical properties. The non-covalent BSA conjugates of these compounds have also been prepared. Compound 2 and the conjugate 2-BSA show extremely high photodynamic activities toward B16 melanoma cancer cell lines. The corresponding 50% growth-inhibitory (IC 50) ratios are 33 and 38 nM, respectively.