Curcumin induces cell death of the main molecular myeloma subtypes, particularly the poor prognosis subgroups

Figures & data

Table 1. HMCLs' characteristics and sensitivity to curcumin

HMCL	HMCLs characteristics			Curcumin
	Translocation	Target genes	TP53	LD50 (μM)
BCN	t(14 ;16)	c-Maf	wt	31.5 ± 1.5
XG6	t(16 ;22)	c-Maf	wt	13.5 ± 5
RPMI 8226	t(14 ;16)	c-Maf	mutated	17.2 ± 3
NAN1	t(14 ;16)	c-Maf	negative **	14.5 ± 0.7
L363	t(20 ;22)	MafB	negative**	17.5 ± 2
MM1S	t(14 ;16)	c-Maf	wt	20.3 ± 1.7
JJN3	t(14 ;16)	c-Maf	negative*	20.8 ± 3.2
ANBL6	t(14 ;16)	c-Maf	truncated	56 ± 4
NCIH929	t(4 ;14)	MMSET/FGFR3	wt	18.4 ± 1
XG7	t(4 ;14)	MMSET	wt	17 ± 2
LP1	t(4 ;14)	MMSET/FGFR3	mutated	17.3 ± 2.3
JIM3	t(4 ;14)	MMSET/FGFR3	mutated	46.6 ± 1.8
OPM2	t(4 ;14)	MMSET/FGFR3	mutated	23.5 ± 1.7
NAN3	t(4 ;14)	MMSET	wt + mutated	15 ± 1
NAN8	t(4 ;14)	MMSET/FGFR3	truncated	15.6 ± 2.5
NAN9	t(4 ;14)	MMSET	wt	17.4 ± 1.9
KMS11	t(4 ;14)	MMSET/FRFR3	negative*	19 ± 0.7
NAN7	t(11 ;14)	CCDN1	truncated	17 ± 3
KMS12PE	t(11 ;14)	CCDN1	mutated	39.5 ± 3.6
XG5	t(11 ;14)	CCDN1	mutated	32.8 ± 3.3
KARPAS 620	t(11 ;14)	CCDN1	mutated	28.9 ± 0.11
SKMM2	t(11 ;14)	CCDN1	mutated	37 ± 2.7
XG1	t(11 ;14)	CCDN1	mutated	17.2 ± 1.4
XG11	t(11 ;14)	CCDN1	mutated	27.8 ± 0.8
U266	t(11 ;14)	CCDN1	mutated	51 ± 5
KMS12BM	t(11 ;14)	CCDN1	mutated	48 ± 3
AMO1	t(12 ;14)	unknown	wt	21.9 ± 2.5
XG2	t(12 ;14)	unknown	mutated	16.4 ± 1.8
KMM1	t(6 ;14)	CCDN3	wt + mutated	20.5 ± 2.3

HMCLs characteristics were previously described.^11,21 LD₅₀ values were determined after 24 h curcumin treatment. Median LD₅₀=20.5 μM

* lack of mRNA expression.

* *lack of protein (premature stop codon. lack of intron splicing).

Figure 1. Curcumin induced cell death of myeloma cells belonging to the main molecular myeloma subgroups. (A) HMCL (n = 29) were treated with curcumin for 24 h. Cell death was measured by FACS analysis of Apo2.7 stained cells. LD₅₀ values were calculated from at least 3 independent experiments. HMCLs were classified according to translocation subtypes. Statistical analysis was performed using Kruskall-Wallis test. Curcumin LD₅₀ of HMCLs were analyzed according to (B) the presence or absence of t(11,14) translocation (Table 1) or (C) TP53 status (Table 1). Statistical analysis was performed using Mann-Whitney test.

Table 2. Sensitivity of primary myeloma cells to curcumin

	Characteristics					Cell death %
Sample	Disease	Status	t(11;14)	t(4;14)	del(17p)	10 μM	20 μM
1	sPCL	R	+	−	−	21%	98%
2	MM	D	−	+	−	nd	87%
3*	sPCL	R	nd	−	−	23%	98%
4	sPCL	R	+	−	+	7%	83%
5	MM	D	−	−	−	85%	94%
6	MM	D	−	−	−	44%	68%
7	MM	R	−	−	−	0%	35%
8	sPCL	R	−	−	+	64%	96%
9	MM	D	−	−	+	52%	98%

MM, multiple myeloma; sPCL, secondary plasma cell leukemia; R, relapse; D, diagnosis; nd, not done. Cells were treated 24 h with curcumin (10 and 20 μM). Cell death was assessed by the loss of CD138 expression. The presence of t(11;14), t(4;14) and del(17p) was assessed by fluorescence in situ hybridization (FISH). *Cells obtained from sample 3 were also treated with melphalan (20 μM), which induced only 13% of cell death.

Figure 2. Primary myeloma cells were killed by curcumin. Primary cells (CD 138⁺) obtained from sample 4 were treated 24 h with the indicated doses of curcumin. Cells were then stained with an anti-CD138-PE mAb. The loss of CD138 staining was representative of cell death. Cell death percentage was calculated relative to untreated cells.

Figure 3. Curcumin cell death was associated with Mcl^-1 decrease and Caspase-3 activation. (A) L363 (highly sensitive) and XG5 (poorly sensitive) cells were treated during 24 h with 15 μM curcumin, cell death was determined by Apo 2.7 staining. Data represent the mean ± SD of 3 independent experiments. (B) Caspase-3 protein levels and activity were determined on cell lysates. Data represent mean ± SD of 3 independent experiments. (C) Cells were treated with curcumin for the indicated times and the expression of Bcl-2 family molecules was assessed by protein gel blotting. Primary purified CD138+ cells (p4 sample) were treated 24 h with 15 μM curcumin, which induced 82% of cell death. (D) Transient knock-down of Mcl-1, Bcl-2 and Bcl-xL was performed on LP1 cells. After 48 h of transfection cells were treated with curcumin for the next 24 h and subjected to Apo 2.7 staining. Efficient silencing of the 3 anti-apoptotic proteins is shown by immunoblotting in the left panel. Data represent mean ± SD of 3 independent experiments. *** = P < 0.001; ** = P < 0.01; * = P < 0.05 and ns = P > 0.05. Statistical analysis was performed using 2-way Anova test.